Methods and materials for scalable 3d cellular redifferentiation

Abstract

Methods and materials for inducing cell redifferentiation of pluripotent stem cells (iPSCs) derived hematopoietic stem cells (iHSCs), including scalable 3D methods using vertical wheel bioreactors.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 685,394, filed August 21, 2024; U.S. Provisional Application No. 63 / 650,039, filed July 29, 2024; and U.S. Provisional Application No. 63 / 539,993, filed September 22, 2023, the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] This invention teaches methods and materials for inducing cell redifferentiation of pluripotent stem cell (iPSC)-derived hematopoietic stem cells (e.g., iHSCs), particularly for generating gamma delta (γδ) T cells derived from such iPSCs. The methods and materials of this invention comprise a scalable three-dimensional (3D) approach to redifferentiating iHSCs using a vertical wheel bioreactor to manufacture iPSC-derived cell therapy products. Background Technology

[0003] Immunotherapy, including T-cell immunotherapy (such as autologous chimeric antigen receptor (CAR) T-cell therapy), is reportedly an effective treatment for some diseases, including some cancers, including some hematologic malignancies. However, applying cell therapy to treat multiple cancer types has encountered many different challenges (see, for example, Advances and challenges of CAR T therapy and suitability of animal models (Review), by Xavier E. Ramos-Cardona, Weichuan Luo, and Sulma I. Mohammed; published online July 12, 2022). Summary of the Invention

[0004] Advances in T-cell immunotherapy, such as autologous CAR T-cell therapy, have been reported; however, unmet challenges remain in the application of cell therapy to treat various cancer types. In this context, the present invention addresses many challenges hindering the acquisition of therapeutic cell products, such as excessively high manufacturing costs, increased complexity, and a lack of consistent and scalable manufacturing processes. For example, the inventors of the present invention utilize iPSCs, particularly capable of self-renewal, gene editing, and multi-lineage differentiation, to advance off-the-shelf, allogeneic, CAR-engineered T-cell therapy platforms with more commercially viable, automated, energy-efficient, and resource-saving manufacturing processes. For instance, the iPSC-derived cell therapy platform according to the present invention is based on the production of γδ T cells, which possess inherent anti-tumor activity, and whose tumor infiltration is associated with better prognosis. Therefore, there is a need in the art for a method to efficiently redifferentiate iPSCs derived from γδ T cells into iPSC-derived HSCs (iHSCs), which serve as a source for producing a sufficient number of robust allogeneic γδ T cells. These iHSCs possess sufficient purity and fold expansion, and exhibit high T cell redifferentiation potential for use in T cell-based products, as do γδ T cells, which possess excellent increased purity and fold expansion properties and are easier to prepare and use. This invention relates to methods and materials for such improvements.

[0005] The inventors have addressed the need in the art for a scalable method to generate iHSCs from iPSCs using a 3D vertical wheel (3D-VW) bioreactor without the use of, for example, serum or other cells (e.g., feeder cells or stromal cells). This method produces iHSCs with high purity, viability, fold expansion, and the potential to redifferentiate into immune effector cells, including lymphocytes such as γδ T cells, particularly iPSC-derived γδ (iγδ) T cells. These unexpected results meet or exceed estimates of yield, purity, viability, fold expansion, and redifferentiation potential based on research-scale 2D culture systems. This disclosure addresses these and other needs.

[0006] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in tissue culture dishes under serum-free and feeder-free culture conditions. The method comprises: seeding human γδ T cell-derived iPSCs in tissue culture dishes including vertical wheels, wherein the iPSCs are maintained for up to approximately 7 days; redifferentiating the iPSCs into iHSCs by timely addition of key growth factors and small molecules and culturing in tissue culture dishes including vertical wheels for approximately 8 days; retaining all cells in the culture on day 8 of culture while removing the culture medium for approximately 2 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on approximately day 10 of culture.

[0007] In some implementations, tissue culture dishes have non-traditional bioreactor geometries.

[0008] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[0009] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ® The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0010] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish including the vertical wheel on days 2, 4, and 6 of a culture lasting approximately 8 days. The step of replacing the used culture medium includes: removing the used culture medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter and adding wash medium at a pump flow rate of 30 mL / min. This wash medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ®Differentiation medium; replace approximately 3 L to approximately 3.5 L of wash medium in the bioreactor and remove 0.1 L of wash medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter; and add approximately 50 mL of wash medium and approximately 50 mL of culture medium to the tissue culture dish including the vertical wheel.

[0011] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0012] In some implementations, the step of preserving all cells includes preserving clusters, half-clusters, and single cells.

[0013] In some embodiments, the step of retaining all cells includes removing used culture medium from a tissue culture dish including a vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium contains advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fresh culture medium does not contain antibiotics.

[0014] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

[0015] In some implementation schemes, the harvesting steps include:

[0016] a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0017] b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium;

[0018] c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension.

[0019] d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the combined cell suspension. ® Differentiation culture medium;

[0020] e) Centrifuge the combined cell suspension to produce a third cell precipitate;

[0021] f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension;

[0022] g) Mix the fourth cell suspension until the cell clusters are no longer visible;

[0023] h) If cell clusters are visible after the mixing step, optionally pass a fourth cell suspension through a cell filter with a 70 µm mesh size to obtain iHSCs; and

[0024] i) Centrifuge the fourth cell suspension.

[0025] In some implementations, the cells are cultured under normoxic conditions.

[0026] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0027] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0028] In some implementations, determining the purity of redifferentiated iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or equivalent methods.

[0029] In some implementations, pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0030] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in tissue culture dishes under serum-free and feeder-free culture conditions, the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on about the 10th day of culture.

[0031] In some implementations, tissue culture dishes have non-traditional bioreactor geometries.

[0032] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[0033] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ® The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0034] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish including the vertical wheel on each of days 2, 4, 6, and 8 of a culture lasting approximately 8 days. The step of replacing the used culture medium includes: a) allowing the cell aggregates to settle in the tissue culture dish including the vertical wheel for approximately 3 to 5 minutes; b) collecting 80% of the used culture medium from the tissue culture dish including the vertical wheel; c) centrifuging the collected culture medium to obtain a cell pellet; and d) resuspending the cell pellet in StemFit. ® The cell suspension is obtained by adding the cell suspension and fresh culture medium to a tissue culture dish containing a vertical wheel.

[0035] In some implementations, the step of replacing the used culture medium on days 2, 4, and 6 of culture includes: i) allowing the cell aggregates to settle in a tissue culture dish including a vertical wheel for approximately 3 to 5 minutes; collecting 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuging the collected culture medium to obtain a cell pellet; and resuspending the cell pellet in StemFit. ® The cell suspension is obtained by adding the cell suspension and fresh culture medium to a tissue culture dish including a vertical wheel, or ii) allowing the cell aggregates to settle in the tissue culture dish including the vertical wheel for about 3 to about 5 minutes; collecting 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuging the collected culture medium to obtain a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit medium. ® The first cell pellet, resuspended in differentiation medium, was added to a tissue culture dish including a vertical wheel; the supernatant was centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet was resuspended in StemFit medium. ® The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to a tissue culture dish containing a vertical wheel.

[0036] In some embodiments, the fresh culture medium added on the second day of culture comprises: i) advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and L-glutamine and GlutaMax. ™ One of the supplements, or ii) StemPro at a ratio of 500:13. ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of the media contains VEGF, bFGF and SCF in a ratio of 1:0.625:0.625, and the second culture medium does not contain antibiotics.

[0037] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0038] In some implementations, the steps of harvesting redifferentiated iHSCs include retaining clusters, hemiclusters, and single cells.

[0039] In some implementations, the harvesting step includes retaining all cells in a tissue culture dish containing a vertical wheel.

[0040] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

[0041] In some implementations, the steps for harvesting redifferentiated iHSCs include:

[0042] a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0043] b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant;

[0044] c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics;

[0045] d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension;

[0046] e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension.

[0047] f) Centrifuge the first cell suspension to produce a third cell precipitate;

[0048] g) The third cell pellet was resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics was added to produce a second cell suspension.

[0049] h) Mix the second cell suspension until the cell clusters are no longer visible;

[0050] i) If cell clusters are visible after the mixing step, optionally pass the second cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[0051] j) Centrifuge the second cell suspension.

[0052] In some implementations, the cells are cultured under normoxic conditions.

[0053] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0054] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0055] In some implementations, determining the purity of redifferentiated iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or equivalent methods.

[0056] In some implementations, pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0057] In one aspect, this document provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, the iHSCs being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), the method comprising the steps of any of the methods described herein.

[0058] In one aspect, this article provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; retaining all cells in the culture on the 8th day of culture while removing the culture medium from the culture for about 2 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on the about 10th day of culture.

[0059] In some implementations, tissue culture dishes have non-traditional bioreactor geometries.

[0060] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[0061] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ® The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0062] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish including the vertical wheel on days 2, 4, and 6 of a culture lasting approximately 8 days. The step of replacing the used culture medium includes: removing the used culture medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter and adding wash medium at a pump flow rate of 30 mL / min. This wash medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium; replace approximately 3 L to approximately 3.5 L of wash medium in the bioreactor and remove 0.1 L of wash medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter; and add approximately 50 mL of wash medium and approximately 50 mL of culture medium to the tissue culture dish including the vertical wheel.

[0063] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0064] In some implementations, the step of preserving all cells includes preserving clusters, half-clusters, and single cells.

[0065] In some embodiments, the step of retaining all cells includes removing used culture medium from a tissue culture dish including a vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium contains advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™One of them, in which the fresh culture medium does not contain antibiotics.

[0066] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

[0067] In some implementation schemes, the harvesting steps include:

[0068] a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0069] b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium;

[0070] c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension.

[0071] d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the combined cell suspension. ® Differentiation culture medium;

[0072] e) Centrifuge the combined cell suspension to produce a third cell precipitate;

[0073] f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension;

[0074] g) Mix the fourth cell suspension until the cell clusters are no longer visible;

[0075] h) If cell clusters are visible after the mixing step, optionally pass a fourth cell suspension through a cell filter with a 70 µm mesh size to obtain iHSCs; and

[0076] i) Centrifuge the fourth cell suspension.

[0077] In some implementations, the cells are cultured under normoxic conditions.

[0078] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0079] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0080] In some implementations, determining the purity of redifferentiated iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or equivalent methods.

[0081] In some implementations, pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0082] In one aspect, this article provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on about the 10th day of culture.

[0083] In some implementations, tissue culture dishes have non-traditional bioreactor geometries.

[0084] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[0085] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ®The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0086] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish including the vertical wheel on each of days 2, 4, 6, and 8 of a culture lasting approximately 8 days. The step of replacing the used culture medium includes: a) allowing the cell aggregates to settle in the tissue culture dish including the vertical wheel for approximately 3 to 5 minutes; b) collecting 80% of the used culture medium from the tissue culture dish including the vertical wheel; c) centrifuging the collected culture medium to obtain a cell pellet; and d) resuspending the cell pellet in StemFit. ® The cell suspension is obtained by adding the cell suspension and fresh culture medium to a tissue culture dish containing a vertical wheel.

[0087] In some implementations, the steps of replacing the used culture medium on days 2, 4, and 6 of cultivation include:

[0088] i) Allow the cell aggregates to settle in a tissue culture dish containing vertical wheels for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish containing vertical wheels; centrifuge the collected culture medium to obtain a cell pellet; resuspend the cell pellet in StemFit. ® The cells are then added to a differentiation medium to obtain a cell suspension; and the cell suspension and fresh medium are added to a tissue culture dish including a vertical wheel, or...

[0089] ii) Allow the cell aggregates to settle in a tissue culture dish including a vertical wheel for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a first cell pellet and a first supernatant; resuspend the first cell pellet in StemFit. ® The first cell pellet, resuspended in differentiation medium, was added to a tissue culture dish including a vertical wheel; the supernatant was centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet was resuspended in StemFit medium. ® The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to a tissue culture dish containing a vertical wheel.

[0090] In some implementations, the fresh culture medium added on the second day of culture contains:

[0091] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[0092] ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them,

[0093] VEGF, bFGF, and SCF were present in a ratio of 1:0.625:0.625, and

[0094] The second culture medium does not contain antibiotics.

[0095] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0096] In some implementations, the steps of harvesting redifferentiated iHSCs include retaining clusters, hemiclusters, and single cells.

[0097] In some embodiments, the step of retaining all cells includes removing used culture medium from a tissue culture dish including a vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium contains advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™One of them, in which the fresh culture medium does not contain antibiotics.

[0098] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

[0099] In some implementations, the steps for harvesting redifferentiated iHSCs include:

[0100] a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0101] b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant;

[0102] c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics;

[0103] d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension;

[0104] e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension.

[0105] f) Centrifuge the first cell suspension to produce a third cell precipitate;

[0106] g) The third cell pellet was resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics was added to produce a second cell suspension.

[0107] h) Mix the second cell suspension until the cell clusters are no longer visible;

[0108] i) If cell clusters are visible after the mixing step, optionally pass the second cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[0109] j) Centrifuge the second cell suspension.

[0110] In some implementations, the cells are cultured under normoxic conditions.

[0111] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0112] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0113] In some implementations, one or more cells have the potential to be highly pure, viable, multiply, and redifferentiated into immune effector cells, including iPSC-derived γδ (iγδ) T cells.

[0114] In some implementations, the step of determining the purity of iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent method, optionally all of them.

[0115] In some implementations, pedigree markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0116] In one aspect, this document provides a composition comprising one or more cells according to any one of the embodiments described herein.

[0117] In one aspect, this document provides the use of one or more cells according to any one of the embodiments described herein in the preparation of cells for treating lesions, diseases, in the preparation of lymphocytes, in bioreactors, in in vitro drug screening for tissue engineering or diseases.

[0118] In one aspect, this document provides a system for performing a method for de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising the steps of any embodiment described herein, wherein a vertical wheel is perpendicular or not perpendicular to the vessel.

[0119] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in a tissue culture dish under serum-free and feeder-free culture conditions. The method includes: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to approximately 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for approximately 8 days; apparatus for retaining all cells in the culture on day 8 of culture while removing the culture medium from the culture for approximately 2 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on approximately day 10 of culture.

[0120] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the method comprising: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on about the 10th day of culture.

[0121] In one aspect, this document provides an iHSC generated according to any of the methods described herein.

[0122] In one aspect, this document provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for about 8 days; apparatus for retaining all cells in the culture on the 8th day of culture while removing the culture medium from the culture for about 2 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on the about 10th day of culture.

[0123] In one aspect, this document provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for about 8 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on about the 10th day of culture.

[0124] In one aspect, this article provides a method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[0125] i) Obtaining one or more human induced pluripotent stem cells (iPSCs), wherein the method further includes one or more of the following steps:

[0126] a) In a tissue culture dish including a vertical wheel, incubate iPSCs in a first culture medium at a stirring speed of about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days. After incubation, remove the entire cell volume from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume.

[0127] The first culture medium contains StemFit ® The first medium contains one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and Rho kinase inhibitor (ROCK inhibitor), and a mixture of Crman 1, Enrica, polyamine, and trans-ISRIB (CEPT), wherein the first medium does not contain antibiotics.

[0128] b) Centrifuge the first cell suspension to produce a first cell precipitate;

[0129] c) The first cell pellet is resuspended in a second culture medium to form a second cell suspension, wherein the second culture medium comprises:

[0130] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ®The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[0131] ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them,

[0132] VEGF, bFGF and SCF were present in a ratio of 1:0.625:0.625, and the second culture medium did not contain antibiotics.

[0133] d) Culture the second cell suspension in a tissue culture dish including a vertical wheel and stir the second cell suspension at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0134] e) Stop stirring for about 5 minutes, during which the cell aggregates settle in the second cell suspension;

[0135] f) Collect approximately 80% of the top volume of the second cell suspension from the tissue culture dish including the vertical wheel;

[0136] g) Centrifuge the collected second cell suspension to form a second cell pellet and resuspend the second cell pellet in approximately 1 mL of StemFit solution. ® Differentiation solution and / or StemPro ™ -34 SFM in one of them;

[0137] h) The resuspended second cell pellet is added to the third culture medium to form a third cell suspension, wherein the third culture medium contains:

[0138] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ®The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0139] ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0140] Furthermore, the third culture medium does not contain antibiotics;

[0141] i) The third cell suspension is placed into the tissue culture dish including a vertical wheel, which contains the remaining second cell suspension, thereby forming a fourth cell suspension;

[0142] j) Culture the fourth cell suspension in a tissue culture dish including a vertical wheel and stir the fourth cell suspension at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0143] k) Stop stirring for about 5 minutes; during this time, cell aggregates will settle in the cell suspension.

[0144] l) Collect approximately 80% of the fourth cell suspension and centrifuge the collected fourth cell suspension to form a third cell pellet, and resuspend the third cell pellet in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM in one of them;

[0145] m) Dissolve in approximately 1 mL of StemFit ® Differentiation solution and StemPro ™ The third cell pellet from one of the -34 SFM cultures is resuspended in a fourth culture medium to form a fifth cell suspension, wherein the fourth culture medium contains:

[0146] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0147] ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0148] Furthermore, the fourth culture medium does not contain antibiotics;

[0149] n) The fifth cell suspension is placed into a tissue culture dish containing the remaining fourth cell suspension, including a vertical wheel, to form the sixth cell suspension;

[0150] o) Culture the sixth cell suspension in a tissue culture dish including a vertical wheel and stir the sixth cell suspension at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0151] p) Repeat steps k) to o); optionally, the sixth cell suspension is centrifuged and the supernatant from the centrifuged sixth cell suspension is centrifuged, and the resulting cell pellet is resuspended in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM, and combined with the seventh cell suspension obtained by resuspending the cell pellet from the first centrifugation to form the eighth cell suspension, the eighth cell suspension is then cultured in a tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days.

[0152] q) Remove all eighth cell suspension from the tissue culture dish including the vertical wheel, and harvest EB and iHSC from the eighth cell suspension by centrifugation to produce fourth cell pellet and first supernatant;

[0153] r) Collect the first supernatant;

[0154] s) The first supernatant is centrifuged to produce a fifth cell pellet, and the fifth cell pellet is resuspended in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium contains advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells in the ninth cell suspension contain iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0155] t) The fourth cell pellet from step q) is resuspended in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension;

[0156] u) Add the fifth culture medium to the tenth cell suspension;

[0157] v) Centrifuge the tenth cell suspension to produce the sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce the eleventh cell suspension.

[0158] w) Add the additional fifth culture medium to the eleventh cell suspension and mix;

[0159] x) Centrifuge the eleventh cell suspension to form the seventh cell pellet, and resuspend the seventh cell pellet in another fifth culture medium to form the twelfth cell suspension.

[0160] y) If cell clumps are present in the twelfth cell suspension, the twelfth cell suspension may optionally be passed through a cell filter with a 70 µm mesh size to obtain iHSCs;

[0161] z) Centrifuge the twelfth cell suspension;

[0162] aa) No serum is introduced in any of the steps a)-z);

[0163] bb) No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-aa).

[0164] cc) Determine the purity of the iHSCs from the first supernatant of step s) and the iHSCs from step y), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and

[0165] dd) to obtain the iHSC generated from scratch.

[0166] In one aspect, this article provides a method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[0167] i) Obtaining one or more human induced pluripotent stem cells (iPSCs), wherein the method further includes one or more of the following steps:

[0168] a) Inoculation of iPSCs, wherein inoculation is performed by suspending iPSCs in a first culture medium in a tissue culture dish including a vertical wheel, thereby producing a first cell suspension, wherein the first culture medium contains StemFit ® Basic 04 medium and StemFit ® One of the following: Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromogen 1, enricane, polyamine and trans-ISRIB (CEPT).

[0169] Furthermore, the first culture medium does not contain antibiotics;

[0170] b) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days, during which embryoid bodies (EB) are generated.

[0171] c) Remove the first culture medium from the tissue culture dish including the vertical wheel and add wash medium at a rate of about 30 mL / min, the wash medium containing advanced DMEM / F12 medium and StemFit® differentiation medium in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1.

[0172] d) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of wash medium in a tissue culture dish including a vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of wash medium from the tissue culture dish including the vertical wheel;

[0173] e) Add approximately 50 mL of wash medium and approximately 50 mL of a second medium to the tissue culture dish including the vertical wheel, wherein the second medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax™ supplements, wherein the second medium does not contain antibiotics;

[0174] f) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0175] g) Remove the second culture medium from the tissue culture dish containing the vertical wheel and add wash medium at a rate of approximately 30 mL / min;

[0176] h) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of wash medium in a tissue culture dish including a vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of wash medium from the tissue culture dish including the vertical wheel.

[0177] i) Add approximately 50 mL of wash medium and approximately 50 mL of third medium to a tissue culture dish including a vertical wheel, wherein the third medium contains advanced DMEM / F12 medium and StemFit medium present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the third culture medium does not contain antibiotics;

[0178] j) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0179] k) Remove the third culture medium from the tissue culture dish containing the vertical wheel and add wash medium at a rate of approximately 30 mL / min;

[0180] l) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of wash medium in a tissue culture dish including a vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of wash medium from the tissue culture dish including the vertical wheel.

[0181] m) Add approximately 50 mL of wash medium and approximately 50 mL of a fourth medium to the tissue culture dish including the vertical wheel, wherein the fourth medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fourth culture medium does not contain antibiotics;

[0182] n) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0183] o) Using a 4µm cell retention filter, at a pump flow rate of approximately 0.69 mL / min (1 L / day), simultaneously remove the fourth culture medium from the tissue culture dish including the vertical wheel and add the fifth culture medium to the tissue culture dish including the vertical wheel within approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days, until 2 L of culture medium has been replaced. The fifth culture medium contains advanced DMEM / F12 medium and StemFit at a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fifth culture medium does not contain antibiotics;

[0184] p) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0185] q) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium;

[0186] r) Add a first volume of 20 mg / mL collagenase II to the first cell pellet, resuspend the first cell pellet, and add a second volume of 20 mg / mL collagenase II to produce a first cell suspension.

[0187] s) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the first cell suspension. ® Differentiation culture medium;

[0188] t) Centrifuge the cell suspension to produce a third cell precipitate;

[0189] u) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors, or antibiotics and StemFit. ® Differentiation culture medium, thereby producing a second cell suspension;

[0190] v) Mix the second cell suspension until the cell clusters are no longer visible;

[0191] w) If cell clusters are visible after the mixing step, the second cell suspension may optionally be passed through a cell filter to obtain iHSC, wherein the cell filter has a 70 µm mesh size;

[0192] x) Centrifuge the second cell suspension;

[0193] y) No serum is introduced in any of the steps a)-x);

[0194] z) No additional cells, including feeder cells or matrix cells, are introduced in any of the steps a)-y).

[0195] aa) Determine the purity of the iHSC from step w), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and

[0196] bb) Obtain the iHSC generated from scratch.

[0197] In one aspect, this article provides a method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[0198] i. Obtaining one or more human induced pluripotent stem cells (iPSCs), wherein the method further comprises one or more of the following steps:

[0199] a. Inoculation of iPSCs, wherein inoculation is carried out by suspending iPSCs in a first culture medium in a bioreactor to produce a first cell suspension, wherein the bioreactor is a vertical wheel bioreactor, and wherein the first culture medium contains StemFit. ® Basic 04 medium and StemFit ® One of the following: Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromogen 1, enricane, polyamine and trans-ISRIB (CEPT).

[0200] Furthermore, the first culture medium does not contain antibiotics;

[0201] b. The first cell suspension is cultured in a bioreactor and stirred at 10 rpm to 60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and stirred for about 1.8 days to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EB) are generated.

[0202] c. Centrifuge the first cell suspension to produce the first cell precipitate;

[0203] d. Resuspend the first cell pellet in a second culture medium to form a second cell suspension, wherein the second culture medium comprises:

[0204] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[0205] ii. StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™-34 nutritional supplements, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, as well as L-glutamine and GlutaMax ™ One of them,

[0206] Furthermore, the second culture medium does not contain antibiotics;

[0207] e. The second cell suspension is cultured and stirred in a bioreactor for approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days;

[0208] f. Stop stirring for about 5 minutes, during which the cell aggregates settle in the second cell suspension;

[0209] g. Collect approximately 80% of the top volume of the second cell suspension from the bioreactor;

[0210] h. Centrifuge the collected second cell suspension to form a second cell pellet, and resuspend the second cell pellet in approximately 1 mL of StemFit solution. ® Differentiation solution and StemPro ™ -34 SFM in one of them;

[0211] i. In approximately 1 mL of StemFit ® Differentiation solution and / or StemPro ™ The second cell pellet in one of the -34 SFM media is resuspended in a third culture medium to form a third cell suspension, wherein the third culture medium contains:

[0212] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0213] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™-34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0214] Furthermore, the third culture medium does not contain antibiotics;

[0215] j. The third cell suspension is placed into the bioreactor containing the remaining second cell suspension to form a fourth cell suspension;

[0216] k. The fourth cell suspension is cultured and stirred in a bioreactor for approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days;

[0217] 1. Stop stirring for about 5 minutes; during this time, cell aggregates will settle in the cell suspension.

[0218] m. Collect approximately 80% of the fourth cell suspension and centrifuge the collected fourth cell suspension to form a third cell pellet, and resuspend the third cell pellet in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM in one of them;

[0219] n. Dissolve in approximately 1 mL of StemFit ® Differentiation solution and StemPro ™ The third cell pellet from one of the -34 SFM cultures is resuspended in a fourth culture medium to form a fifth cell suspension, wherein the fourth culture medium contains:

[0220] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0221] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™-34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0222] Furthermore, the fourth culture medium does not contain antibiotics;

[0223] o. The fifth cell suspension is placed into a bioreactor containing the remaining fourth cell suspension to form the sixth cell suspension;

[0224] p. The sixth cell suspension is cultured and stirred in a bioreactor for approximately 1.8 to approximately 2.2 days, preferably approximately 2 days;

[0225] q. Repeat steps l) to p);

[0226] r. Remove all eighth cell suspension from the bioreactor and harvest EB and iHSC from the cell suspension by centrifugation, thereby producing fourth cell precipitate and first supernatant;

[0227] s. Collect the first supernatant;

[0228] t. Centrifuge the first supernatant to produce a fifth cell pellet and resuspend the fifth cell pellet in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium contains advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells in the ninth cell suspension contain iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0229] u. Resuspend the fourth cell pellet from step r) in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension;

[0230] v. Add the fifth culture medium to the tenth cell suspension;

[0231] w. Centrifuge the tenth cell suspension to produce the sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce the eleventh cell suspension;

[0232] x. Add the additional fifth culture medium to the eleventh cell suspension and mix;

[0233] y. Centrifuge the eleventh cell suspension to form the seventh cell pellet, and resuspend the seventh cell pellet in another fifth culture medium to form the twelfth cell suspension;

[0234] z. Pass the twelfth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70µm mesh size;

[0235] aa. Determine the purity of the iHSC from the first supernatant of step t) and the iHSC from step z), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0236] bb. No serum is introduced in any of the steps a)-aa).

[0237] cc. No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-bb); and

[0238] dd. Obtain the iHSC generated from scratch.

[0239] In some implementations, EB and γδ T cell-derived iPSCs are derived from tissue culture dishes.

[0240] In some implementations, the first cell suspension, the second cell suspension, the fourth cell suspension, the sixth cell suspension, and / or the eighth cell suspension are cultured under normoxic conditions.

[0241] In some implementations, the method further includes further purification and / or separation of iHSCs.

[0242] In some implementations, the obtained iHSCs are isolated redifferentiated cells or cells that can be further purified and / or isolated.

[0243] In some embodiments, the method further includes determining all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or an equivalent method.

[0244] In some implementations, any of steps a)-cc) includes gently dispersing the cells using a pipette or its equivalent.

[0245] In some embodiments, the concentration of cells in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 50 mL to about 70 mL.

[0246] In some implementations, the concentration of cells in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 60 mL.

[0247] In some implementations, the culture medium in step a) also contains a ROCK inhibitor.

[0248] In some implementations, the culture medium in step a) further comprises a mixture of chroma1, enricane, polyamine, and trans-ISRIB (CEPT).

[0249] In some implementations, after step b), all cell volumes of the first cell suspension are removed from the bioreactor before centrifugation.

[0250] In some implementations, cell morphology is imaged via microscopy, such as brightfield microscopy.

[0251] In some implementations, the culture medium in steps d), i), n), or t) contains L-glutamine.

[0252] In some implementations, the culture medium in steps d), i), n), or t) contains GlutaMax. ™ .

[0253] In some implementations, the culture medium in step i) or n) contains TPO.

[0254] In some implementations, the culture medium in step i) or n) contains EPO.

[0255] In some implementations, centrifugation in any step is performed at 100g for 1 minute at brake position 6.

[0256] In some implementations, centrifugation at 300g for 5 minutes is performed in any step.

[0257] In some implementations, centrifugation at 300g for 3 minutes is performed in any step.

[0258] In some implementations, the centrifugation in step r) is performed twice: first at brake position 6 with 100g for 1 minute, and then at 300g for 5 minutes.

[0259] In some implementations, the tenth cell suspension of step u) is incubated with collagenase at 37°C for 20 minutes.

[0260] In some implementations, the eleventh cell suspension from step w) is incubated with TrypLE at 37°C for 5 minutes.

[0261] In some embodiments, passing the twelfth cell suspension through a second cell filter having a 40µm mesh size is further included in step z).

[0262] In some implementations, iPSC cells, EB cells, and iHSC cells are cultured at 37°C and 5% CO2.

[0263] In some implementations, the bioreactor wheel rotates at 60 rpm.

[0264] In some implementations, the bioreactor wheel rotates at 30 rpm.

[0265] In some implementations, the bioreactor wheel rotates at 24 rpm.

[0266] In some implementations, the bioreactor wheel rotates at 15 rpm.

[0267] In some implementations, the bioreactor wheel is perpendicular to the bioreactor vessel.

[0268] In some implementations, the bioreactor wheel is not perpendicular to the bioreactor vessel.

[0269] In some embodiments, the concentration of iPSCs in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 90 mL to about 110 mL.

[0270] In some implementations, the concentration of iPSCs in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 100 mL.

[0271] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 0.45L to about 0.55L.

[0272] In some implementations, the concentration of iPSCs in the first culture medium is approximately 20,000 cells / mL in a volume of approximately 0.5 L.

[0273] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 2L to about 5L.

[0274] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 10L to about 20L.

[0275] In one aspect, this article provides one or more cells obtained from a method of de novo generation of iHSCs under serum-free and feeder-free culture conditions, wherein said method includes:

[0276] i. Obtain at least one human iPSC clone, and

[0277] The method further includes one or more of the following steps:

[0278] a. Redifferentiating the at least one human iPSC clone by culturing it in a first cell culture medium to produce a first cell suspension, wherein the first cell culture medium contains StemFit supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF. ® Basic 04 medium and StemFit ® One of the Basic 03 culture media, wherein the first cell culture medium further comprises CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromium 1, enricane, polyamine and trans-ISRIB (CEPT), wherein the culture is carried out in a vertical wheel bioreactor at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0279] b. Centrifuge the first cell suspension containing iPSCs from step a) to form a first precipitate and resuspend the first precipitate in a second cell culture medium to form a second cell suspension, wherein the second cell culture medium comprises:

[0280] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, which also contains L-glutamine and GlutaMax. ™ One of them, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or

[0281] ii. StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™-34 nutritional supplements, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, as well as L-glutamine and GlutaMax ™ One of them,

[0282] Furthermore, the second cell culture medium does not contain antibiotics;

[0283] c. In a vertical wheel bioreactor, a second cell suspension containing iHSCs is cultured at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0284] d. Stop stirring the vertical wheel bioreactor for about 5 minutes and collect about 80% of the second cell suspension;

[0285] e. Centrifuge 80% of the second cell suspension to produce a second cell precipitate;

[0286] f. The second precipitate is suspended in a third cell culture medium to form a third cell suspension, wherein the third cell culture medium contains:

[0287] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0288] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0289] Furthermore, the third culture medium does not contain antibiotics, and the third cell suspension is placed in a vertical wheel bioreactor containing the remaining second cell suspension to form a fourth cell suspension;

[0290] g. The fourth cell suspension is cultured again in a vertical wheel bioreactor for about 1.8 days to about 2.2 days, preferably about 2 days;

[0291] h. Stop the vertical wheel bioreactor for approximately 5 minutes;

[0292] i. Collect approximately 80% of the fourth cell suspension and centrifuge approximately 80% of the fourth suspension to produce a third cell precipitate;

[0293] j. The third cell pellet is resuspended in a fourth cell culture medium to form a fifth cell suspension, wherein the fourth cell culture medium contains:

[0294] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0295] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0296] Furthermore, the fifth cell suspension is added to the remaining fourth cell suspension in the vertical wheel bioreactor to produce the sixth cell suspension;

[0297] k. Culture the sixth cell suspension in a vertical wheel bioreactor for approximately 1.8 to approximately 2.2 days, preferably approximately 2 days;

[0298] l. Repeat steps h) to k);

[0299] m. Centrifuge the eighth cell suspension to produce the first supernatant and the fourth cell precipitate;

[0300] n. Collect and centrifuge the first supernatant to produce a fifth cell pellet and resuspend the fifth cell pellet in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium contains advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells in the ninth cell suspension contain iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0301] o. Resuspend the fourth cell pellet from step m) in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension;

[0302] p. Add the fifth culture medium to the tenth cell suspension;

[0303] q. Centrifuge the tenth cell suspension to produce the sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce the eleventh cell suspension;

[0304] r. Add the additional fifth culture medium to the eleventh cell suspension and mix;

[0305] s. Centrifuge the eleventh cell suspension to form the seventh cell pellet, and resuspend the seventh cell pellet in another fifth culture medium to form the twelfth cell suspension;

[0306] t. Pass the twelfth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70µm mesh size;

[0307] u. Determine the purity of iHSCs from the first supernatant of step n) and iHSCs from step t), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0308] v. No serum is introduced in any of the steps a)-u);

[0309] w. No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-v); and

[0310] x. Obtain the iHSC generated from scratch.

[0311] In some implementations, one or more cells have the potential to be highly pure, viable, multiply, and redifferentiated into immune effector cells, including iPSC-derived γδ (iγδ) T cells.

[0312] In some implementations, EB and γδ T cell-derived iPSCs are derived from tissue culture dishes.

[0313] In some implementations, the first cell suspension, the second cell suspension, the fourth cell suspension, the sixth cell suspension, and / or the eighth cell suspension are cultured under normoxic conditions.

[0314] In some implementations, the method further includes further purification and / or separation of iHSCs.

[0315] In some implementations, the obtained iHSCs are isolated redifferentiated cells or cells that can be further purified and / or isolated.

[0316] In some embodiments, the method further includes determining all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or an equivalent method.

[0317] In some implementations, any of steps a)-w) includes gently dispersing the cells using a pipette or its equivalent.

[0318] In some embodiments, the concentration of cells in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 50 mL to about 70 mL.

[0319] In some implementations, the concentration of cells in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 60 mL.

[0320] In some implementations, the culture medium in step a) also contains a ROCK inhibitor.

[0321] In one embodiment, the culture medium in step a) further comprises a mixture of chroma1, enricane, polyamine, and trans-ISRIB (CEPT).

[0322] In some implementations, after step a), all cell volumes of the first cell suspension are removed from the bioreactor before centrifugation.

[0323] In some implementations, cell morphology is imaged via microscopy, such as brightfield microscopy.

[0324] In some implementations, the culture medium in steps b), f), j), or n) contains L-glutamine.

[0325] In some implementations, the culture medium in steps b), f), j), or n) contains GlutaMax. ™ .

[0326] In some implementations, the culture medium in step f) or j) contains TPO.

[0327] In some implementations, the culture medium in step f) or j) contains EPO.

[0328] In some implementations, centrifugation in any step is performed at 100g for 1 minute at brake position 6.

[0329] In some implementations, centrifugation at 300g for 5 minutes is performed in any step.

[0330] In some implementations, centrifugation at 300g for 3 minutes is performed in any step.

[0331] In some implementations, the centrifugation in step l) is performed twice: first at brake position 6 with 100g for 1 minute, and then at 300g for 5 minutes.

[0332] In some implementations, the cell suspension from step o) is incubated with collagenase at 37°C for 20 minutes.

[0333] In some implementations, the eleventh cell suspension from step q) is incubated with TrypLE at 37°C for 5 minutes.

[0334] In some embodiments, passing the twelfth cell suspension through a second cell filter having a 40µm mesh size is further included in step t).

[0335] In some implementations, iPSC cells, EB cells, and iHSC cells are cultured at 37°C and 5% CO2.

[0336] In some implementations, the bioreactor wheel rotates at 60 rpm.

[0337] In some implementations, the bioreactor wheel rotates at 30 rpm.

[0338] In some implementations, the bioreactor wheel rotates at 24 rpm.

[0339] In some implementations, the bioreactor wheel rotates at 15 rpm.

[0340] In some implementations, the bioreactor wheel is perpendicular to the bioreactor vessel.

[0341] In some implementations, the bioreactor wheel is not perpendicular to the bioreactor vessel.

[0342] In some embodiments, the concentration of iPSCs in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 90 mL to about 110 mL.

[0343] In some implementations, the concentration of iPSCs in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 100 mL.

[0344] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 0.45 L to about 0.55 L.

[0345] In some implementations, the concentration of iPSCs in the first culture medium is approximately 20,000 cells / mL in a volume of approximately 0.5 L.

[0346] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 2L to about 5L.

[0347] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 10L to about 20L.

[0348] In one aspect, this document provides a composition comprising one or more cells as described herein.

[0349] In one aspect, this article provides the use of one or more of the cells described herein in the preparation of cells for treating diseases, in the preparation of lymphocytes, in bioreactors, and in in vitro drug screening for tissue engineering or diseases. Attached Figure Description

[0350] Figures 1A to 1B The iHSC redifferentiation process is illustrated. Figure 1A The process flow of the iHSC redifferentiation process is shown. Figure 1BA schematic diagram of an exemplary bioreactor system of the present invention is shown, including a waste component, a culture medium reservoir component, a pump component, a reactor chamber component (black circle) including a vertical wheel component, a cell inlet component (not shown), a cell release component (not shown), and a connector component. Adapted from Bioreactor Systems for Bone Tissue Engineering, Article in Tissue Engineering Part B Reviews, 2011, DOI: 10.1089 / ten.TEB.2010.0612.

[0351] Figure 2 The stepwise redifferentiation process of iHSCs in a 3D-VW bioreactor is shown.

[0352] Figure 3 The purity, total yield, viability, and fold expansion of iHSCs redifferentiated from single human γδ T cell-derived iPSC clones in a 3D-VW bioreactor culture are illustrated, where iHSCs were defined as lineage marker-negative CD34+ cells. Both thrombopoietin (TPO) and erythropoietin (EPO) supported iHSC redifferentiation. iHSCs were detected as single cells and in dissociated cell clusters. This novel 3D iHSC protocol meets exemplary acceptance criteria for iHSC+ purity.

[0353] Figure 4 The 3D Vertical Wheel (VW) method is demonstrated to reproducibly generate iHSCs with high purity, yield, and sufficient fold expansion. In both study-scale 2D and 3D-VW culture systems, iHSCs were redifferentiated from two different iPSC clones. An initial exemplary success criterion for iHSC differentiation was 70% iHSC cells (Lin-CD34+) and 0.5-fold iHSC expansion. For both iPSC clones, purity and cell viability were comparable between the 3D and 2D methods.

[0354] Figure 5 This study demonstrates that iHSCs generated using the 3D-VW method possess the potential for γδ T cell redifferentiation. iHSCs generated in the 3D-VW method produced iγδ T cells with the same purity as iHSCs generated using the 2D method. The redifferentiation of iHSCs into iγδ T cells was carried out in a 2D culture system according to a basic protocol, utilizing iHSCs redifferentiated in both the 2D and 3D culture systems. The 3D-derived iHSCs used in this experiment were generated by seeding iPSCs at a 1.5× standard protocol seeding density. The initial exemplary success criteria for iγδ T cell redifferentiation were 20% to 40% iγδ T cells (CD3+TCRγδ+) and a 5-fold expansion.

[0355] In summary, these data indicate that the 3D-VW method generates iHSC cells with T cell differentiation potential.

[0356] Figure 6 Historical data on the T cell differentiation potential of multiple clones used for iCD34 redifferentiation in an exemplary 3D-VW reactor are shown, as measured in terms of purity, fold expansion, and yield. For dissociated single cells, a 50% harvested single-cell population was treated with the same enzymes used for dissociation clusters to examine the effect of such enzymes on T cell differentiation potential. The initial exemplary success criterion for iHSC differentiation was 70% iHSC cells (defined as Lin-CD34). + ) and 0.5-fold iHSC amplification. Three independent cell clones, namely clone B, clone C, and clone E, possess high T cell differentiation potential. Detailed Implementation

[0357] definition

[0358] Various terms relating to various aspects of the specification are used throughout the specification and claims. Unless otherwise specified, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in accordance with the definitions provided herein.

[0359] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” and “described” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, and so on.

[0360] As used herein, the term "about," when referring to measurable values ​​such as quantity, time interval, etc., means to cover a variation of at most ±10% from the specified value, as such variation is suitable for performing the methods disclosed in this invention. Unless otherwise specified, all figures used in the specification and claims to represent the quantity of components, characteristics such as molecular weight, reaction conditions, etc., should in all cases be understood to be modified by the term "about." Therefore, unless stated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values, which may vary according to the desired characteristics sought to be obtained according to the invention. To a minimum and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should at least be interpreted according to the significant digits of the reported value and by applying customary rounding.

[0361] Although the numerical ranges and parameters used to illustrate the broad scope of the invention are approximate, the values ​​presented in specific embodiments are reported as precisely as possible. However, any numerical value inherently contains some error, which will necessarily arise from the standard deviation present in its respective test measurement method.

[0362] The term "treatment" refers to any success or indication of success achieved in reducing or improving an injury, lesion, or condition, including any objective or subjective parameters such as symptom reduction, relief, weakening, or increased patient tolerance of the condition; slowing the rate of degeneration or decline; reducing the degree of failure at the degenerative endpoint; improving the physical or mental health of the subject; or prolonging survival. Treatment can be assessed through objective or subjective parameters, including the results of physical examination, neurological examination, or psychiatric evaluation.

[0363] The term "induced pluripotent stem cell" (hereinafter referred to as "iPSC") means a stem cell that has pluripotency, allowing it to differentiate into any cell type present in a living organism, and also possesses the ability to proliferate (i.e., self-renew). It encompasses any cell capable of redifferentiating into hematopoietic stem cells (HSCs) to be used in this invention. iPSCs are preferably derived from primates (e.g., monkeys, orangutans, chimpanzees, humans), more preferably humans.

[0364] The term "iHSC" refers to hematopoietic stem cells derived from iPSCs.

[0365] The terms "non-traditional geometry" or "non-traditional bioreactor" refer to those geometries or bioreactors that are not part of the "traditional geometry" or "traditional bioreactor," i.e., those geometries that predate the geometry of this invention. "Non-traditional geometry" or "non-traditional bioreactor" can refer to a geometry or bioreactor that combines radial and axial flow components. A vertical wheel bioreactor is a non-limiting example of a non-traditional bioreactor. "Traditional geometry" or "traditional bioreactor" can refer to a stirred tank geometry or a stirred tank bioreactor, respectively. "Traditional geometry" or "traditional bioreactor" can refer to a geometry or bioreactor having radial flow components. Horizontal blade bioreactors and turbine impellers are non-limiting examples of conventional bioreactors. The terms “conventional geometry” and “conventional bioreactor” are further defined in Borys, BS, et al., “Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirredsuspension bioreactors.” Stem Cell Research & Therapy. 2021, 12:55, the full text of which is incorporated herein by reference. The terms “3D vertical wheel bioreactor” and “tissue culture dish including vertical wheels” are used interchangeably herein.

[0366] The implementation schemes described herein are not limited to specific methods, reagents, compounds, compositions, or biological systems, which can of course vary.

[0367] Methods for generating iHSC

[0368] γδ T cells were chosen as the origin of iPSCs because the epigenetic and transcriptional memory of these initiating cells are known to influence and enhance the redifferentiation potential of iPSCs to this cell type. An optimized three-dimensional (3D) serum-free, feeder-free (SFFF) protocol was used to redifferentiate γδ T-iPSCs into iHSCs, and then into iγδ T cells, as shown below. Figure 1A , Figure 1B and Figure 2As shown. The purity and fold expansion of redifferentiated iPSC clones into iHSCs and iHSCs into iγδ T cells were determined. Cell images were recorded. Based on results from the 3D and 2D redifferentiation protocols, exemplary success criteria for the purity and fold expansion of redifferentiated iHSCs and iγδ T cells were established. The 3D-VW bioreaction protocol used to redifferentiate iPSCs into iHSCs produced high purity, high cell viability, high yield, and sufficient fold expansion, as shown. Figures 3 to 6 As shown. This protocol also yielded iγδ T cells with purity meeting the exemplary success criteria and fold expansion consistent with iHSCs generated using the 2D method. These data demonstrate the efficiency and effectiveness of the 3D-VW bioreaction protocol for generating iHSCs from γδ T-iPSCs, as... Figures 3 to 6 As shown in the image.

[0369] Maintain iPSC before initiating redifferentiation

[0370] As is known in the art, iPSC colonies typically exhibit circular colonies with densely packed cells, a high nucleus-to-cytoplasm ratio, well-defined edges, and distinct boundaries.

[0371] iPSCs can be thawed from a cryopreservation solution, and a desired number of live cells can be used in the methods described herein. Cell counting, size, and viability determination can be performed by any method known in the art, for example, using a NucleoCounter NC-200. Live cells with a preferred size (e.g., 10 μm to 25 μm) can be used in the steps described herein.

[0372] iPSC can be 1.0×10 5 Up to 1.8×10 5 Seed at the appropriate cell density. After seeding, maintain iPSCs in suitable tissue or cell culture dishes, such as, but not limited to, tissue culture dishes with vertical wheels. Incubate the tissue or cell culture dishes at 37°C and 5% CO2.

[0373] iPSC in StemFit ® Growth was carried out in Basic 04 medium, which also contained basic fibroblast growth factor (bFGF) at a final concentration of approximately 90 ng / mL to approximately 110 ng / mL bFGF, preferably approximately 100 ng / mL bFGF. This medium contained 10 µM Y-27632 ROCK inhibitor or a mixture of chromogen 1, enricane, polyamine, and trans-ISRIB (CEPT). StemFit ®Basic 04 Cell Culture Medium (Ajinomoto) is a defined-component, animal-free culture medium for the culture of human pluripotent stem cells. This medium is a complete medium and requires no additional supplements.

[0374] StemFit ® Basic 03 cell culture medium (Ajinomoto) is a defined-component, animal-free culture medium for the culture of human pluripotent stem cells. StemPro ™ -34 serum-free medium (SFM) (Gibco) ™ StemPro is a serum-free culture medium specially formulated to support the development of artificial blood cells (including HSCs and progenitor cells isolated from bone marrow, neonatal cord blood, and peripheral blood) in culture. ™ -34 SFM is a flexible cell culture medium that can be used in a variety of applications.

[0375] During the maintenance period, the cell morphology of iPSCs can be examined at regular intervals (e.g., daily). Cell morphology can be determined by microscopy, such as bright-field microscopy. Once the cells become bright and have well-defined boundaries, the colonies can redifferentiate into iHSCs as discussed herein. As is known in the art, the culture medium can be changed at regular intervals.

[0376] iPSCs were redifferentiated into iHSCs using a small-volume bioreactor including vertical wheels.

[0377] This invention provides a method for redifferentiating iPSCs into iHSCs using a small-volume bioreactor or tissue culture dish including vertical wheels. Non-limiting exemplary volumes include 0.1 L bioreactors or tissue culture dishes.

[0378] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in tissue culture dishes under serum-free and feeder-free culture conditions, the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on about the 10th day of culture.

[0379] In some implementations, tissue culture dishes have non-traditional bioreactor geometries as discussed herein.

[0380] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶.5 Seed at a density of cells / mL.

[0381] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ® The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0382] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish including the vertical wheel on each of days 2, 4, 6, and 8 of a culture lasting approximately 8 days. The step of replacing the used culture medium includes: a) allowing the cell aggregates to settle in the tissue culture dish including the vertical wheel for approximately 3 to 5 minutes; b) collecting 80% of the used culture medium from the tissue culture dish including the vertical wheel; c) centrifuging the collected culture medium to obtain a cell pellet; and d) resuspending the cell pellet in StemFit. ® The cell suspension is obtained by adding the cell suspension and fresh culture medium to a tissue culture dish containing a vertical wheel.

[0383] In some implementations, the step of replacing the used culture medium on days 2, 4, and 6 of culture includes: i) allowing the cell aggregates to settle in a tissue culture dish including a vertical wheel for approximately 3 to 5 minutes; collecting 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuging the collected culture medium to obtain a cell pellet; and resuspending the cell pellet in StemFit. ® The cell suspension is obtained by adding the cell suspension and fresh culture medium to a tissue culture dish including a vertical wheel, or ii) allowing the cell aggregates to settle in the tissue culture dish including the vertical wheel for about 3 to about 5 minutes; collecting 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuging the collected culture medium to obtain a first cell pellet and a first supernatant; resuspending the first cell pellet in StemFit medium. ® The first cell pellet, resuspended in differentiation medium, was added to a tissue culture dish including a vertical wheel; the supernatant was centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet was resuspended in StemFit medium. ®The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to a tissue culture dish containing a vertical wheel.

[0384] In some implementations, the fresh culture medium added on the second day of culture contains:

[0385] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[0386] ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them,

[0387] VEGF, bFGF, and SCF were present in a ratio of 1:0.625:0.625, and

[0388] The second culture medium does not contain antibiotics.

[0389] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0390] In some implementations, the steps of harvesting redifferentiated iHSCs include retaining clusters, hemiclusters, and single cells.

[0391] In some implementations, the step of harvesting redifferentiated iHSCs includes retaining all cells in a tissue culture dish containing a vertical wheel.

[0392] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

[0393] In some implementations, the steps for harvesting redifferentiated iHSCs include:

[0394] a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0395] b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant;

[0396] c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics;

[0397] d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension;

[0398] e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension.

[0399] f) Centrifuge the first cell suspension to produce a third cell precipitate;

[0400] g) The third cell pellet was resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics was added to produce a second cell suspension.

[0401] h) Mix the second cell suspension until the cell clusters are no longer visible;

[0402] i) If cell clusters are visible after the mixing step, optionally pass the second cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[0403] j) Centrifuge the second cell suspension.

[0404] In some implementations, the cells are cultured under normoxic conditions.

[0405] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0406] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0407] In some implementations, determining the purity of redifferentiated iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or equivalent methods.

[0408] In some implementations, pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0409] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the method comprising: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on about the 10th day of culture.

[0410] In one aspect, this article provides a method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[0411] i) Obtaining one or more human induced pluripotent stem cells (iPSCs), wherein the method further includes one or more of the following steps:

[0412] a) In a tissue culture dish including a vertical wheel, incubate iPSCs in a first culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, with stirring at about 10 rpm to 60 rpm, preferably 40 rpm. Afterward, remove the entire cell volume from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume.

[0413] The first culture medium contains StemFit ® The first medium contains one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and Rho kinase inhibitor (ROCK inhibitor), and a mixture of Crman 1, Enrica, polyamine, and trans-ISRIB (CEPT), wherein the first medium does not contain antibiotics.

[0414] b) Centrifuge the first cell suspension to produce a first cell precipitate;

[0415] c) The first cell pellet is resuspended in a second culture medium to form a second cell suspension, wherein the second culture medium comprises:

[0416] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[0417] ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them,

[0418] VEGF, bFGF and SCF were present in a ratio of 1:0.625:0.625, and the second culture medium did not contain antibiotics.

[0419] d) Culture the second cell suspension in a tissue culture dish including a vertical wheel and stir the second cell suspension at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0420] e) Stop stirring for about 5 minutes, during which the cell aggregates settle in the second cell suspension;

[0421] f) Collect approximately 80% of the top volume of the second cell suspension from the tissue culture dish including the vertical wheel;

[0422] g) Centrifuge the collected second cell suspension to form a second cell pellet and resuspend the second cell pellet in approximately 1 mL of StemFit solution. ® Differentiation solution and / or StemPro ™ -34 SFM in one of them;

[0423] h) The resuspended second cell pellet is added to the third culture medium to form a third cell suspension, wherein the third culture medium contains:

[0424] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0425] ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0426] Furthermore, the third culture medium does not contain antibiotics;

[0427] i) The third cell suspension is placed into the tissue culture dish including a vertical wheel, which contains the remaining second cell suspension, thereby forming a fourth cell suspension;

[0428] j) Culture the fourth cell suspension in a tissue culture dish including a vertical wheel and stir the fourth cell suspension at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0429] k) Stop stirring for about 5 minutes; during this time, cell aggregates will settle in the cell suspension.

[0430] l) Collect approximately 80% of the fourth cell suspension and centrifuge the collected fourth cell suspension to form a third cell pellet, and resuspend the third cell pellet in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM in one of them;

[0431] m) Dissolve in approximately 1 mL of StemFit ® Differentiation solution and StemPro ™ The third cell pellet from one of the -34 SFM cultures is resuspended in a fourth culture medium to form a fifth cell suspension, wherein the fourth culture medium contains:

[0432] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0433] ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0434] Furthermore, the fourth culture medium does not contain antibiotics;

[0435] n) The fifth cell suspension is placed into a tissue culture dish containing the remaining fourth cell suspension, including a vertical wheel, to form the sixth cell suspension;

[0436] o) Culture the sixth cell suspension in a tissue culture dish including a vertical wheel and stir the sixth cell suspension at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0437] p) Repeat steps k) to o); optionally, the sixth cell suspension is centrifuged and the supernatant from the centrifuged sixth cell suspension is centrifuged, and the resulting cell pellet is resuspended in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM, and combined with the seventh cell suspension obtained by resuspending the cell pellet from the first centrifugation to form the eighth cell suspension, the eighth cell suspension is then cultured in a tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days.

[0438] q) Remove all eighth cell suspension from the tissue culture dish including the vertical wheel, and harvest EB and iHSC from the eighth cell suspension by centrifugation to produce fourth cell pellet and first supernatant;

[0439] r) Collect the first supernatant;

[0440] s) The first supernatant is centrifuged to produce a fifth cell pellet, and the fifth cell pellet is resuspended in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium contains advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells in the ninth cell suspension contain iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0441] t) The fourth cell pellet from step q) is resuspended in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension;

[0442] u) Add the fifth culture medium to the tenth cell suspension;

[0443] v) Centrifuge the tenth cell suspension to produce the sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce the eleventh cell suspension.

[0444] w) Add the additional fifth culture medium to the eleventh cell suspension and mix;

[0445] x) Centrifuge the eleventh cell suspension to form the seventh cell pellet, and resuspend the seventh cell pellet in another fifth culture medium to form the twelfth cell suspension.

[0446] y) If cell clumps are present in the twelfth cell suspension, the twelfth cell suspension may optionally be passed through a cell filter with a 70 µm mesh size to obtain iHSCs;

[0447] z) Centrifuge the twelfth cell suspension;

[0448] aa) No serum is introduced in any of the steps a)-z);

[0449] bb) No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-aa).

[0450] cc) Determine the purity of the iHSCs from the first supernatant of step s) and the iHSCs from step y), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and

[0451] dd) to obtain the iHSC generated from scratch.

[0452] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) in tissue culture dishes under serum-free and feeder-free culture conditions, the iHSCs being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising:

[0453] a) An apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days;

[0454] b) A device for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days;

[0455] c) A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

[0456] In one aspect, this article provides a method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[0457] 1. Obtaining one or more human induced pluripotent stem cells (iPSCs), wherein the method further comprises one or more of the following steps:

[0458] a. Inoculation of iPSCs, wherein inoculation is carried out by suspending iPSCs in a first culture medium in a bioreactor to produce a first cell suspension, wherein the bioreactor is a vertical wheel bioreactor, and wherein the first culture medium contains StemFit. ® Basic 04 medium and StemFit ® One of the following: Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromogen 1, enricane, polyamine and trans-ISRIB (CEPT).

[0459] Furthermore, the first culture medium does not contain antibiotics;

[0460] b. The first cell suspension is cultured in a bioreactor and stirred at 10 rpm to 60 rpm, preferably 40 rpm, wherein the first cell suspension is cultured and stirred for about 1.8 days to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EB) are generated.

[0461] c. Centrifuge the first cell suspension to produce the first cell precipitate;

[0462] d. Resuspend the first cell pellet in a second culture medium to form a second cell suspension, wherein the second culture medium comprises:

[0463] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[0464] ii. StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, as well as L-glutamine and GlutaMax ™ One of them,

[0465] Furthermore, the second culture medium does not contain antibiotics;

[0466] e. The second cell suspension is cultured and stirred in a bioreactor for approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days;

[0467] f. Stop stirring for about 5 minutes, during which the cell aggregates settle in the second cell suspension;

[0468] g. Collect approximately 80% of the top volume of the second cell suspension from the bioreactor;

[0469] h. Centrifuge the collected second cell suspension to form a second cell pellet, and resuspend the second cell pellet in approximately 1 mL of StemFit solution. ® Differentiation solution and StemPro ™ -34 SFM in one of them;

[0470] i. In approximately 1 mL of StemFit ® Differentiation solution and / or StemPro ™ The second cell pellet in one of the -34 SFM media is resuspended in a third culture medium to form a third cell suspension, wherein the third culture medium contains:

[0471] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0472] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0473] Furthermore, the third culture medium does not contain antibiotics;

[0474] j. The third cell suspension is placed into the bioreactor containing the remaining second cell suspension to form a fourth cell suspension;

[0475] k. The fourth cell suspension is cultured and stirred in a bioreactor for approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days;

[0476] 1. Stop stirring for about 5 minutes; during this time, cell aggregates will settle in the cell suspension.

[0477] m. Collect approximately 80% of the fourth cell suspension and centrifuge the collected fourth cell suspension to form a third cell pellet, and resuspend the third cell pellet in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM in one of them;

[0478] n. Dissolve in approximately 1 mL of StemFit ® Differentiation solution and StemPro ™ The third cell pellet from one of the -34 SFM cultures is resuspended in a fourth culture medium to form a fifth cell suspension, wherein the fourth culture medium contains:

[0479] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0480] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0481] Furthermore, the fourth culture medium does not contain antibiotics;

[0482] o. The fifth cell suspension is placed into a bioreactor containing the remaining fourth cell suspension to form the sixth cell suspension;

[0483] p. The sixth cell suspension is cultured and stirred in a bioreactor for approximately 1.8 to approximately 2.2 days, preferably approximately 2 days;

[0484] q. Repeat steps l) to p);

[0485] r. Remove all eighth cell suspension from the bioreactor and harvest EB and iHSC from the cell suspension by centrifugation, thereby producing fourth cell precipitate and first supernatant;

[0486] s. Collect the first supernatant;

[0487] t. Centrifuge the first supernatant to produce a fifth cell pellet and resuspend the fifth cell pellet in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium contains advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells in the ninth cell suspension contain iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0488] u. Resuspend the fourth cell pellet from step r) in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension;

[0489] v. Add the fifth culture medium to the tenth cell suspension;

[0490] w. Centrifuge the tenth cell suspension to produce the sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce the eleventh cell suspension;

[0491] x. Add the additional fifth culture medium to the eleventh cell suspension and mix;

[0492] y. Centrifuge the eleventh cell suspension to form the seventh cell pellet, and resuspend the seventh cell pellet in another fifth culture medium to form the twelfth cell suspension;

[0493] z. Pass the twelfth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70µm mesh size;

[0494] aa. Determine the purity of the iHSC from the first supernatant of step t) and the iHSC from step z), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0495] bb. No serum is introduced in any of the steps a)-aa).

[0496] cc. No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-bb); and

[0497] dd. Obtain the iHSC generated from scratch.

[0498] In the 3D solution, it is possible to operate at 37°C and 5% CO2, while containing StemFit. ®Basic 04 medium or StemFit ® iPSCs are cultured in a first medium of Basic 03 medium and stirred at about 10 to 60 rpm, preferably about 40 rpm, for about 1.8 to about 2.2 days, preferably about two days. This first medium also contains CHIR99021 (glycogen synthase kinase inhibitor), Rho kinase inhibitor (ROCK inhibitor) or Crmon 1, enlicaxate, a mixture of polyamine and trans-ISRIB (CEPT), bone morphogenetic protein 4 (BMP4), and recombinant human vascular endothelial growth factor (rhVEGF). The morphology of redifferentiated iPSCs can be determined by microscopy, such as bright-field microscopy, at the beginning of the culture period and / or at regular intervals. Culture of iPSCs in this medium can induce mesodermal formation, which is a prerequisite for hematopoietic lineage specialization and differentiation.

[0499] Following the mesodermal induction culture period in the 3D-VW bioreactor protocol, iPSC-derived cell intermediates can be collected and subsequently cultured in a secondary medium at 37°C and 5% CO2 with stirring at approximately 10 to 60 rpm, preferably approximately 40 rpm, to initiate hematopoietic differentiation, for approximately 1.8 to approximately 2.2 days, preferably approximately two days. The secondary cell culture medium comprises advanced DMEM / F12 medium (Thermo Fisher Scientific catalog number 12634) and StemFit in a ratio of approximately 3.9:0.9 to approximately 4.1:1.1, preferably approximately 4:1. ® Differentiation medium (Ajinomoto) also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, and stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of them. Alternatively, the second cell culture medium may contain StemPro at a ratio of 500:13. ™ -34 SFM + StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, and also contains SB431542, rhVEGF, bFGF, SCF, as well as L-glutamine and GlutaMax. ™One of them. SB431542 was diluted from a 5 mM stock solution concentration to a final concentration of approximately 2 µM in order to use the minimum amount of dimethyl sulfoxide (DMSO) as a diluent. Cell morphology can be determined at the beginning of the culture period and / or at regular intervals by microscopy, such as bright-field microscopy. Advanced DMEM / F12 medium contains amino acids, vitamins, inorganic salts, proteins, reducing elements, trace elements, dextran, ethanolamine, sodium hypoxanthine, linoleic acid, lipoic acid, phenol red, putrescine 2HCl, sodium pyruvate, and thymidine. StemFit ® The differentiation medium is a chemically defined medium for differentiating human embryonic stem (ES) cells and iPSCs without animal-derived (CD-AOF) supplements.

[0500] Following the culture period used to initiate hematopoietic differentiation in the 3D-VW bioreactor protocol, iPSC-derived cell intermediates can be collected and subsequently cultured in a third medium at 37°C and 5% CO2, with stirring at approximately 10 to 60 rpm, preferably approximately 40 rpm, to induce angiogenic differentiation for approximately 1.8 to approximately 2.2 days, preferably approximately 2 days. The third medium comprises advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to approximately 4.1:1.1, preferably approximately 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), L-glutamine, and GlutaMax. ™ One of the following, and one of thrombopoietin (TPO) and erythropoietin (EPO). Alternatively, the third cell culture medium may contain StemPro at a ratio of 500:13. ™ -34 SFM + StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, and also contains IL-3, IL-6, SCF, rhVEGF, Flt3L, L-glutamine and GlutaMax ™ One of the following, and one of TPO and EPO. TPO was prepared from a stock solution concentration of 100 μg / mL to a final concentration of 30 ng / mL. EPO was prepared from a stock solution concentration of 500 IU / mL to a final concentration of 10 IU / mL. Cell morphology can be determined at the beginning of the culture period and / or at regular intervals by microscopy, such as bright-field microscopy.

[0501] Following the culture period for inducing angiogenesis in the 3D-VW bioreactor protocol, iHSCs can be collected and subsequently cultured in a fourth medium at 37°C and 5% CO2 with stirring at 10 to 60 rpm, preferably about 40 rpm, for about 1.8 to about 4.2 days, preferably about 2 to about 4 days, for the transition phase from endothelial cells to hematopoietic cells. The fourth medium contains advanced DMEM / F12 medium and StemFit in a ratio of about 3.9:0.9 to about 4.1:1.1, preferably about 4:1. ® Differentiation medium, which also contains SCF, IL-6, L-glutamine and GlutaMax. ™ One of the four cell cultures, and one of TPO and EPO. Alternatively, the fourth cell culture medium may contain StemPro at a ratio of 500:13. ™ -34 SFM + StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, and also contains SCF, IL-6, L-glutamine and GlutaMax. ™ One of the following, and one of TPO and EPO. TPO was prepared from a stock solution concentration of 100 μg / mL to a final concentration of 30 ng / mL. EPO was prepared from a stock solution concentration of 500 IU / mL to a final concentration of 10 IU / mL. Cell morphology can be determined at the beginning of the culture period and / or at regular intervals by microscopy, such as bright-field microscopy.

[0502] In the 3D-VW bioreactor protocol, after approximately 7.8 to 8.2 days of redifferentiation, preferably approximately 8 days, non-adhesive iHSCs in the culture medium can be collected, centrifuged, resuspended in a fourth cell culture medium, and then added back to the culture.

[0503] Approximately 10 days after redifferentiation in the 3D-VW bioreactor protocol, the appearance and morphology of iHSCs can be monitored by microscopy, such as bright-field microscopy.

[0504] Single-cell fractions of iHSCs from the 3D-VW bioreactor protocol can be harvested by centrifugation and resuspended in a fifth-cell culture medium containing advanced DMEM / F12 medium but without cytokines or growth factors. iHSCs can then be counted as described herein.

[0505] Cell cluster fractions from iHSCs derived from the 3D-VW bioreactor protocol can be resuspended in type II collagenase and fifth-generation cell culture medium, followed by centrifugation and resuspending in TrypLE and fifth-generation cell culture medium, and then further centrifugation and resuspending in fifth-generation cell culture medium. The dispersed cells can then be passed through a cell filter with a 70 µm mesh size. Cell counting can be performed as described herein.

[0506] After harvesting two iHSC fractions from the 3D-VW bioreactor protocol in the fifth culture medium, cell purity can be assessed, for example, using flow cytometry or equivalent methods to analyze the presence or absence of certain markers. Purity can be defined by the absence of expression of one or more lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a, and the presence of expression via CD34 (CD34+). In other words, the harvested cells are lineage marker negative and CD34+. Following purity analysis, the redifferentiated iHSCs can be cryopreserved.

[0507] In some implementations, EB and γδ T cell-derived iPSCs are derived from tissue culture dishes.

[0508] In some implementations, any cell suspension is cultured under normoxic conditions.

[0509] In some implementations, the method further includes further purification and / or separation of iHSCs.

[0510] In some implementations, the obtained iHSCs are isolated redifferentiated cells or cells that can be further purified and / or isolated.

[0511] In some embodiments, the method further includes determining all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or an equivalent method.

[0512] In some implementations, any step includes gently dispersing the cells using a pipette or its equivalent.

[0513] In some embodiments, the concentration of cells in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 50 mL to about 70 mL.

[0514] In some implementations, the concentration of cells in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 60 mL.

[0515] In some implementations, the culture medium in step a) also contains a ROCK inhibitor.

[0516] In some implementations, the culture medium in step a) further comprises a mixture of chroma1, enricane, polyamine, and trans-ISRIB (CEPT).

[0517] In some implementations, after step b), all cell volumes of the first cell suspension are removed from the bioreactor before centrifugation.

[0518] In some implementations, cell morphology is imaged via microscopy, such as brightfield microscopy.

[0519] In some implementations, the culture medium in any step contains L-glutamine.

[0520] In some implementations, the culture medium in any step contains GlutaMax. ™ .

[0521] In some implementations, the culture medium in any step contains TPO.

[0522] In some implementations, the culture medium in any step contains EPO.

[0523] In some implementations, centrifugation in any step is performed at 100g for 1 minute at brake position 6.

[0524] In some implementations, centrifugation at 300g for 5 minutes is performed in any step.

[0525] In some implementations, centrifugation at 300g for 3 minutes is performed in any step.

[0526] In some implementations, centrifugation at any step is performed twice: first at brake position 6 with 100g for 1 minute, and then at 300g for 5 minutes.

[0527] In some implementations, the tenth cell suspension is incubated with collagenase at 37°C for 20 minutes.

[0528] In some implementations, the eleventh cell suspension is incubated with TrypLE at 37°C for 5 minutes.

[0529] In some embodiments, passing the twelfth cell suspension through a second cell filter having a sieve size of 40 µm is also included.

[0530] In some implementations, iPSC cells, EB cells, and iHSC cells are cultured at 37°C and 5% CO2.

[0531] In some implementations, the bioreactor wheel rotates at 60 rpm.

[0532] In some implementations, the bioreactor wheel rotates at 30 rpm.

[0533] In some implementations, the bioreactor wheel rotates at 24 rpm.

[0534] In some implementations, the bioreactor wheel rotates at 15 rpm.

[0535] In some implementations, the bioreactor wheel is perpendicular to the bioreactor vessel.

[0536] In some implementations, the bioreactor wheel is not perpendicular to the bioreactor vessel.

[0537] In some embodiments, the concentration of iPSCs in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 90 mL to about 110 mL.

[0538] In some implementations, the concentration of iPSCs in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 100 mL.

[0539] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 0.45L to about 0.55L.

[0540] In some implementations, the concentration of iPSCs in the first culture medium is approximately 20,000 cells / mL in a volume of approximately 0.5 L.

[0541] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 2L to about 5L.

[0542] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 10L to about 20L.

[0543] iPSCs were redifferentiated into iHSCs using a large-volume bioreactor including vertical wheels.

[0544] This invention provides a method for redifferentiating iPSCs into iHSCs using a large-volume bioreactor or tissue culture dish including vertical wheels. Non-limiting exemplary volumes include 3L bioreactors or tissue culture dishes.

[0545] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in tissue culture dishes under serum-free and feeder-free culture conditions, the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; retaining all cells in the culture on the 8th day of culture while removing the culture medium from the culture for about 2 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on the approximately 10th day of culture.

[0546] In some implementations, tissue culture dishes have non-traditional bioreactor geometries.

[0547] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[0548] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ® The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0549] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish including the vertical wheel on days 2, 4, and 6 of a culture lasting approximately 8 days. The step of replacing the used culture medium includes: removing the used culture medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter and adding wash medium at a pump flow rate of 30 mL / min. This wash medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ®Differentiation medium; replace approximately 3 L to approximately 3.5 L of wash medium in the bioreactor and remove 0.1 L of wash medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter; and add approximately 50 mL of wash medium and approximately 50 mL of culture medium to the tissue culture dish including the vertical wheel.

[0550] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0551] In some implementations, the step of preserving all cells includes preserving clusters, half-clusters, and single cells.

[0552] In some embodiments, the step of retaining all cells includes removing used culture medium from a tissue culture dish including a vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium contains advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fresh culture medium does not contain antibiotics.

[0553] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

[0554] In some implementation schemes, the harvesting steps include:

[0555] a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0556] b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium;

[0557] c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension.

[0558] d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the combined cell suspension. ® Differentiation culture medium;

[0559] e) Centrifuge the combined cell suspension to produce a third cell precipitate;

[0560] f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension;

[0561] g) Mix the fourth cell suspension until the cell clusters are no longer visible;

[0562] h) If cell clusters are visible after the mixing step, optionally pass a fourth cell suspension through a cell filter with a 70 µm mesh size to obtain iHSCs; and

[0563] i) Centrifuge the fourth cell suspension.

[0564] In one aspect, this article provides a method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[0565] i) Obtaining one or more human induced pluripotent stem cells (iPSCs), wherein the method further includes one or more of the following steps:

[0566] a) Inoculation of iPSCs, wherein inoculation is performed by suspending iPSCs in a first culture medium in a tissue culture dish including a vertical wheel, thereby producing a first cell suspension, wherein the first culture medium contains StemFit ® Basic 04 medium and StemFit ® One of the following: Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromogen 1, enricane, polyamine and trans-ISRIB (CEPT).

[0567] Furthermore, the first culture medium does not contain antibiotics;

[0568] b) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days, during which embryoid bodies (EB) are generated.

[0569] c) Remove the first culture medium from the tissue culture dish including the vertical wheel and add wash medium at a rate of about 30 mL / min. This wash medium contains advanced DMEM / F12 medium and StemFit in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation culture medium;

[0570] d) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of wash medium in a tissue culture dish including a vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of wash medium from the tissue culture dish including the vertical wheel;

[0571] e) Add approximately 50 mL of wash medium and approximately 50 mL of a second medium to the tissue culture dish including the vertical wheel, wherein the second medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax™ supplements, wherein the second medium does not contain antibiotics;

[0572] f) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0573] g) Remove the second culture medium from the tissue culture dish containing the vertical wheel and add wash medium at a rate of approximately 30 mL / min;

[0574] h) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of wash medium in a tissue culture dish including a vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of wash medium from the tissue culture dish including the vertical wheel.

[0575] i) Add approximately 50 mL of wash medium and approximately 50 mL of third medium to a tissue culture dish including a vertical wheel, wherein the third medium contains advanced DMEM / F12 medium and StemFit medium present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the third culture medium does not contain antibiotics;

[0576] j) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0577] k) Remove the third culture medium from the tissue culture dish containing the vertical wheel and add wash medium at a rate of approximately 30 mL / min;

[0578] l) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of wash medium in a tissue culture dish including a vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of wash medium from the tissue culture dish including the vertical wheel.

[0579] m) Add approximately 50 mL of wash medium and approximately 50 mL of a fourth medium to the tissue culture dish including the vertical wheel, wherein the fourth medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fourth culture medium does not contain antibiotics;

[0580] n) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0581] o) Using a 4µm cell retention filter, at a pump flow rate of approximately 0.69 mL / min (1 L / day), simultaneously remove the fourth culture medium from the tissue culture dish including the vertical wheel and add the fifth culture medium to the tissue culture dish including the vertical wheel within approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days, until 2 L of culture medium has been replaced. The fifth culture medium contains advanced DMEM / F12 medium and StemFit at a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fifth culture medium does not contain antibiotics;

[0582] p) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0583] q) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium;

[0584] r) Add a first volume of 20 mg / mL collagenase II to the first cell pellet, resuspend the first cell pellet, and add a second volume of 20 mg / mL collagenase II to produce a first cell suspension.

[0585] s) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the first cell suspension. ® Differentiation culture medium;

[0586] t) Centrifuge the cell suspension to produce a third cell precipitate;

[0587] u) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors, or antibiotics and StemFit. ® Differentiation culture medium, thereby producing a second cell suspension;

[0588] v) Mix the second cell suspension until the cell clusters are no longer visible;

[0589] w) If cell clusters are visible after the mixing step, the second cell suspension may optionally be passed through a cell filter to obtain iHSC, wherein the cell filter has a 70 µm mesh size;

[0590] x) Centrifuge the second cell suspension;

[0591] y) No serum is introduced in any of the steps a)-x);

[0592] z) No additional cells, including feeder cells or matrix cells, are introduced in any of the steps a)-y).

[0593] aa) Determine the purity of the iHSC from step w), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and

[0594] bb) Obtain the iHSC generated from scratch.

[0595] In some implementations, the cells are cultured under normoxic conditions.

[0596] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0597] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0598] In some implementations, determining the purity of redifferentiated iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or equivalent methods.

[0599] In some implementations, pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0600] In one aspect, this article provides a method for de novo generation of hematopoietic stem cells (iHSCs) derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs) in a tissue culture dish under serum-free and feeder-free culture conditions. The method includes: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to approximately 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for approximately 8 days; apparatus for retaining all cells in the culture on day 8 of culture while removing the culture medium from the culture for approximately 2 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on approximately day 10 of culture.

[0601] Cells produced by the method

[0602] In one aspect, this article provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; retaining all cells in the culture on the 8th day of culture while removing the culture medium from the culture for about 2 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on the about 10th day of culture.

[0603] In some implementations, tissue culture dishes have non-traditional bioreactor geometries.

[0604] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[0605] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ®The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0606] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish including the vertical wheel on days 2, 4, and 6 of a culture lasting approximately 8 days. The step of replacing the used culture medium includes: removing the used culture medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter and adding wash medium at a pump flow rate of 30 mL / min. This wash medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium; replace approximately 3 L to approximately 3.5 L of wash medium in the bioreactor and remove 0.1 L of wash medium from the tissue culture dish including the vertical wheel using a 40 µm cell retention filter; and add approximately 50 mL of wash medium and approximately 50 mL of culture medium to the tissue culture dish including the vertical wheel.

[0607] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0608] In some implementations, the step of preserving all cells includes preserving clusters, half-clusters, and single cells.

[0609] In some embodiments, the step of retaining all cells includes removing used culture medium from a tissue culture dish including a vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium contains advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fresh culture medium does not contain antibiotics.

[0610] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

[0611] In some implementation schemes, the harvesting steps include:

[0612] a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0613] b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium;

[0614] c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension.

[0615] d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the combined cell suspension. ® Differentiation culture medium;

[0616] e) Centrifuge the combined cell suspension to produce a third cell precipitate;

[0617] f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension;

[0618] g) Mix the fourth cell suspension until the cell clusters are no longer visible;

[0619] h) If cell clusters are visible after the mixing step, optionally pass a fourth cell suspension through a cell filter with a 70 µm mesh size to obtain iHSCs; and

[0620] i) Centrifuge the fourth cell suspension.

[0621] In some implementations, the cells are cultured under normoxic conditions.

[0622] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0623] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0624] In some implementations, determining the purity of redifferentiated iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or equivalent methods.

[0625] In some implementations, pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0626] In one aspect, this article provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising: seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; redifferentiating the iPSCs into iHSCs by culturing them in the tissue culture dish including the vertical wheel for about 8 days; and harvesting the redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, on about the 10th day of culture.

[0627] In some implementations, tissue culture dishes have non-traditional bioreactor geometries.

[0628] In some implementations, iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[0629] In some embodiments, the tissue culture dish including the vertical wheel also includes a first culture medium containing StemFit. ® The basic 04 medium contains one of the following: CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Cr1, Enrica, polyamine and trans-ISRIB (CEPT), wherein BMP4 and rhVEGF are present in a 1:1 ratio, and wherein the first medium does not contain antibiotics.

[0630] In some embodiments, the method further includes replacing the used culture medium in the tissue culture dish, including the vertical wheel, on each of the 2nd, 4th, 6th, and 8th days of culture, which lasts for approximately 8 days. The step of replacing the used culture medium includes:

[0631] a) Allow the cell aggregates to settle in the tissue culture dish, including the vertical wheel, for approximately 3 to 5 minutes;

[0632] b) Collect 80% of the used culture medium from the tissue culture dish including the vertical wheel;

[0633] c) Centrifuge the collected culture medium to obtain a cell pellet;

[0634] d) Resuspend the cell pellet in StemFit. ® In a differentiation culture medium, a cell suspension is obtained; and

[0635] e) Add the cell suspension and fresh culture medium to the tissue culture dish containing the vertical wheel.

[0636] In some implementations, the steps of replacing the used culture medium on days 2, 4, and 6 of cultivation include:

[0637] i) Allow the cell aggregates to settle in a tissue culture dish containing vertical wheels for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish containing vertical wheels; centrifuge the collected culture medium to obtain a cell pellet; resuspend the cell pellet in StemFit. ® The cells are then added to a differentiation medium to obtain a cell suspension; and the cell suspension and fresh medium are added to a tissue culture dish including a vertical wheel, or...

[0638] ii) Allow the cell aggregates to settle in a tissue culture dish including a vertical wheel for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a first cell pellet and a first supernatant; resuspend the first cell pellet in StemFit. ® The first cell pellet, resuspended in differentiation medium, was added to a tissue culture dish including a vertical wheel; the supernatant was centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet was resuspended in StemFit medium. ® The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to a tissue culture dish containing a vertical wheel.

[0639] In some embodiments, the fresh culture medium added on the second day of culture comprises: i) advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation culture medium, and also containing SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax™ supplements, or ii) StemPro at a ratio of 500:13. ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of the media contains VEGF, bFGF and SCF in a ratio of 1:0.625:0.625, and the second culture medium does not contain antibiotics.

[0640] In some implementations, iPSCs dissociate into single cells on day 7 of culture.

[0641] In some implementations, the steps of harvesting redifferentiated iHSCs include retaining clusters, hemiclusters, and single cells.

[0642] In some implementations, the harvesting step includes retaining all cells in a tissue culture dish containing a vertical wheel.

[0643] In some implementations, the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

[0644] In some implementations, the steps for harvesting redifferentiated iHSCs include:

[0645] a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet.

[0646] b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant;

[0647] c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics;

[0648] d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension;

[0649] e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension.

[0650] f) Centrifuge the first cell suspension to produce a third cell precipitate;

[0651] g) The third cell pellet was resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics was added to produce a second cell suspension.

[0652] h) Mix the second cell suspension until the cell clusters are no longer visible;

[0653] i) If cell clusters are visible after the mixing step, optionally pass the second cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[0654] j) Centrifuge the second cell suspension.

[0655] In some implementations, the cells are cultured under normoxic conditions.

[0656] In some implementations, the method further includes purification and / or separation of redifferentiated iHSCs.

[0657] In some implementations, the redifferentiated iHSCs are isolated differentiated cells or cells that can be further purified and / or isolated.

[0658] In some implementations, one or more cells have the potential to be highly pure, viable, multiply, and redifferentiated into immune effector cells, including iPSC-derived γδ (iγδ) T cells.

[0659] In some implementations, the step of determining the purity of iHSCs includes determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a via flow cytometry or an equivalent method, optionally all of them.

[0660] In some implementations, pedigree markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[0661] In one aspect, this document provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for about 8 days; apparatus for retaining all cells in the culture on the 8th day of culture while removing the culture medium from the culture for about 2 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on the about 10th day of culture.

[0662] In one aspect, this document provides one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; apparatus for redifferentiating the iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for about 8 days; and apparatus for harvesting the redifferentiated iHSCs into two different cell fractions, namely a single-cell fraction and a multi-cell fraction, on about the 10th day of culture.

[0663] In one aspect, this article provides one or more cells obtained from a method of de novo generation of iHSCs under serum-free and feeder-free culture conditions, wherein said method includes:

[0664] i. Obtain at least one human iPSC clone, and

[0665] The method further includes one or more of the following steps:

[0666] a. Redifferentiating the at least one human iPSC clone by culturing it in a first cell culture medium to produce a first cell suspension, wherein the first cell culture medium contains StemFit supplemented with basic fibroblast growth factor (bFGF) to a final concentration of approximately 100 ng / mL bFGF. ® Basic 04 medium and StemFit ®One of the Basic 03 culture media, wherein the first cell culture medium further comprises CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromium 1, enricane, polyamine and trans-ISRIB (CEPT), wherein the culture is carried out in a vertical wheel bioreactor at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0667] b. Centrifuge the first cell suspension containing iPSCs from step a) to form a first precipitate and resuspend the first precipitate in a second cell culture medium to form a second cell suspension, wherein the second cell culture medium comprises:

[0668] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, which also contains L-glutamine and GlutaMax. ™ One of them, SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, bFGF, and stem cell factor (SCF), or

[0669] ii. StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, as well as L-glutamine and GlutaMax ™ One of them,

[0670] Furthermore, the second cell culture medium does not contain antibiotics;

[0671] c. In a vertical wheel bioreactor, a second cell suspension containing iHSCs is cultured at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[0672] d. Stop stirring the vertical wheel bioreactor for about 5 minutes and collect about 80% of the second cell suspension;

[0673] e. Centrifuge 80% of the second cell suspension to produce a second cell precipitate;

[0674] f. The second precipitate is suspended in a third cell culture medium to form a third cell suspension, wherein the third cell culture medium contains:

[0675] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-6 (IL-6), rhVEGF, IL-3, one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[0676] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0677] Furthermore, the third culture medium does not contain antibiotics, and the third cell suspension is placed in a vertical wheel bioreactor containing the remaining second cell suspension to form a fourth cell suspension;

[0678] g. The fourth cell suspension is cultured again in a vertical wheel bioreactor for about 1.8 days to about 2.2 days, preferably about 2 days;

[0679] h. Stop the vertical wheel bioreactor for approximately 5 minutes;

[0680] i. Collect approximately 80% of the fourth cell suspension and centrifuge approximately 80% of the fourth suspension to produce a third cell precipitate;

[0681] j. The third cell pellet is resuspended in a fourth cell culture medium to form a fifth cell suspension, wherein the fourth cell culture medium contains:

[0682] i. Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax.™ One of them, or

[0683] ii. StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[0684] Furthermore, the fifth cell suspension is added to the remaining fourth cell suspension in the vertical wheel bioreactor to produce the sixth cell suspension;

[0685] k. Culture the sixth cell suspension in a vertical wheel bioreactor for approximately 1.8 to approximately 2.2 days, preferably approximately 2 days;

[0686] l. Repeat steps h) to k);

[0687] m. Centrifuge the eighth cell suspension to produce the first supernatant and the fourth cell precipitate;

[0688] n. Collect and centrifuge the first supernatant to produce a fifth cell pellet and resuspend the fifth cell pellet in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium contains advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells in the ninth cell suspension contain iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0689] o. Resuspend the fourth cell pellet from step m) in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension;

[0690] p. Add the fifth culture medium to the tenth cell suspension;

[0691] q. Centrifuge the tenth cell suspension to produce the sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce the eleventh cell suspension;

[0692] r. Add the additional fifth culture medium to the eleventh cell suspension and mix;

[0693] s. Centrifuge the eleventh cell suspension to form the seventh cell pellet, and resuspend the seventh cell pellet in another fifth culture medium to form the twelfth cell suspension;

[0694] t. Pass the twelfth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70µm mesh size;

[0695] u. Determine the purity of iHSCs from the first supernatant of step n) and iHSCs from step t), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[0696] v. No serum is introduced in any of the steps a)-u);

[0697] w. No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-v); and

[0698] x. Obtain the iHSC generated from scratch.

[0699] In some implementations, one or more cells have the potential to be highly pure, viable, multiply, and redifferentiated into immune effector cells, including iPSC-derived γδ (iγδ) T cells.

[0700] In some implementations, EB and γδ T cell-derived iPSCs are derived from tissue culture dishes.

[0701] In some implementations, the first cell suspension, the second cell suspension, the fourth cell suspension, the sixth cell suspension, and / or the eighth cell suspension are cultured under normoxic conditions.

[0702] In some implementations, the method further includes further purification and / or separation of iHSCs.

[0703] In some implementations, the obtained iHSCs are isolated redifferentiated cells or cells that can be further purified and / or isolated.

[0704] In some embodiments, the method further includes determining all of the lineage markers CD3, CD19, CD14, CD11b, CD56, CD20, CD16, CD2, and CD235a by flow cytometry or an equivalent method.

[0705] In some implementations, any step includes gently dispersing the cells using a pipette or its equivalent.

[0706] In some embodiments, the concentration of cells in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 50 mL to about 70 mL.

[0707] In some implementations, the concentration of cells in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 60 mL.

[0708] In some implementations, the culture medium in step a) also contains a ROCK inhibitor.

[0709] In some implementations, the culture medium in step a) further comprises a mixture of chroma1, enricane, polyamine, and trans-ISRIB (CEPT).

[0710] In some implementations, after step a), all cell volumes of the first cell suspension are removed from the bioreactor before centrifugation.

[0711] In some implementations, cell morphology is imaged via microscopy, such as brightfield microscopy.

[0712] In some implementations, the culture medium in any step contains L-glutamine.

[0713] In some implementations, the culture medium in any step contains GlutaMax. ™ .

[0714] In some implementations, the culture medium in any step contains TPO.

[0715] In some implementations, the culture medium in any step contains EPO.

[0716] In some implementations, centrifugation in any step is performed at 100g for 1 minute at brake position 6.

[0717] In some implementations, centrifugation at 300g for 5 minutes is performed in any step.

[0718] In some implementations, centrifugation at 300g for 3 minutes is performed in any step.

[0719] In some implementations, centrifugation in any step is performed twice: first at brake position 6 with 100g for 1 minute, and then at 300g for 5 minutes.

[0720] In some implementations, the tenth cell suspension is incubated with collagenase at 37°C for 20 minutes.

[0721] In some implementations, the eleventh cell suspension is incubated with TrypLE at 37°C for 5 minutes.

[0722] In some embodiments, passing the twelfth cell suspension through a second cell filter having a sieve size of 40 µm is also included.

[0723] In some implementations, iPSC cells, EB cells, and iHSC cells are cultured at 37°C and 5% CO2.

[0724] In some implementations, the bioreactor wheel rotates at 60 rpm.

[0725] In some implementations, the bioreactor wheel rotates at 30 rpm.

[0726] In some implementations, the bioreactor wheel rotates at 24 rpm.

[0727] In some implementations, the bioreactor wheel rotates at 15 rpm.

[0728] In some implementations, the bioreactor wheel is perpendicular to the bioreactor vessel.

[0729] In some implementations, the bioreactor wheel is not perpendicular to the bioreactor vessel.

[0730] In some embodiments, the concentration of iPSCs in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 90 mL to about 110 mL.

[0731] In some implementations, the concentration of iPSCs in the first culture medium is approximately 150,000 cells / mL in a volume of approximately 100 mL.

[0732] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 0.45 L to about 0.55 L.

[0733] In some implementations, the concentration of iPSCs in the first culture medium is approximately 20,000 cells / mL in a volume of approximately 0.5 L.

[0734] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 2L to about 5L.

[0735] In some embodiments, the concentration of iPSCs in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 10L to about 20L.

[0736] In one aspect, this document provides a composition comprising one or more cells as described herein.

[0737] In one aspect, this article provides the use of one or more of the cells described herein in the preparation of cells for treating diseases, in the preparation of lymphocytes, in bioreactors, and in in vitro drug screening for tissue engineering or diseases.

[0738] Reduced cell viability due to enzymatic dissociation of single-cell hPSCs has been reported. Therefore, hPSCs typically grow as colonies or aggregates, and clump seeding is used as a seeding strategy for bioreactor culture. This strategy imposes limitations on scalability, such as difficulty in controlling clumping size, leading to apoptosis and differentiation due to uneven aggregate size. Studies using single-cell seeding as a seeding strategy in conventional bioreactors require high cell seeding densities and produce low cell yields. Surprisingly, the invention presented herein overcomes these challenges by maintaining single cells throughout the process described herein using, for example, a cell retention filter that retains them instead of removing them. It has been unexpectedly found that the presence of single cells, and thus the heterogeneous environment composed of single cells and cell aggregates, advances such processes, benefiting both cell yield and quality.

[0739] Therefore, this invention positively impacts bioecosystems and the environment by driving advancements that help reduce human resources, electricity, energy consumption, carbon emissions, and overall environmental impact. The invention offers features of automation and efficiency, providing more efficient methods and systems that reduce the need for large amounts of manual labor while minimizing the environmental impact associated with resource-intensive activities. The automation of this invention optimizes energy consumption, simplifies operations, and reduces waste, resulting in increased efficiency and reduced carbon emissions. This invention enables more sustainable production, including the development of cleaner and more resource-efficient technologies and the adoption of circular economy practices such as recycling and waste reduction, as it minimizes material waste (including biomass loss), optimizes supply chains, and implements greener manufacturing practices. This invention thus brings about disruptive changes that drive resource efficiency, reduce waste and related carbon emissions, and promote sustainability.

[0740] 3D Vertical Wheel Bioreactor

[0741] As described herein, iPSCs suspended in culture medium were cultured in a 3D vertical wheel bioreactor, also referred to herein as a tissue culture dish incorporating vertical wheels. Not wishing to be bound by theory, the vertical wheel bioreactor was used to combine radial and axial flow components to produce cells via rotation of the vertical wheel impeller within a U-shaped vessel. This allowed for the suspension and gentle agitation of the cells. The use of vertical wheel bioreactors is detailed in Croughan, MS et al., Initial power measurements for a family of novel vertical-wheel bioreactors. Can. J. Chem. Eng. 2022, 1-12; Dang, T. et al., Computational fluiddynamic characterization of vertical-wheel bioreactors used for effective scale-up of human induced pluripotent stem cell aggregate culture. Can. J. Chem. Eng. 2021, 1-18; Borys, BS et al., Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel-stirred suspension bioreactors. Stem Cell Research & Therapy. 2021, 12:55; and Cuesta-Gomez, N. et al., Suspension culture improves iPSC expansion and pluripotency phenotype. Stem Cell Research & Therapy. 2023, 14:154; All of these references are incorporated herein by reference in their full text. The adaptation of the 3D vertical wheel bioreactor to the redifferentiation of iPSC-derived intermediates is surprising, as it has so far been primarily used for iPSC maintenance and has faced challenges in its application. The hydrodynamics of the 3D vertical wheel bioreactor differs from that of a typical stirred tank bioreactor because it provides reduced cell shear.

[0742] As those skilled in the art will understand, the dimensions of the components of a vertical wheel bioreactor, including a vertical wheel impeller and a U-shaped vessel, can vary. In some embodiments, a 0.5L bioreactor may use a vertical wheel impeller with a diameter of about 73 mm and a width of about 25 mm, and a U-shaped vessel with a radius of about 43 mm. The impeller radius may be about 85% of the radius of the U-shaped vessel. In some embodiments, a 3L bioreactor may use a vertical wheel impeller with a diameter of about 137 mm and a width of about 37 mm, and a U-shaped vessel with a radius of about 83 mm. The impeller radius may be about 83% of the radius of the U-shaped vessel. In some embodiments, a 15L bioreactor may use a vertical wheel impeller with a diameter of about 227 mm and a width of about 113 mm, and a U-shaped vessel with a radius of about 138 mm. The impeller radius may be about 83% of the radius of the U-shaped vessel.

[0743] Computational fluid dynamics modeling supports the use of models across scales by generating scale-up equations and calculating volume-averaged hydrodynamic values. The k-ε turbulence model is a commonly used model for simulating flow characteristics in suspended bioreactors. This model uses the following two equations to represent the conservation of momentum and mass:

[0744]

[0745]

[0746] Where ρ is density, u is the Cartesian velocity vector, t is time, P is pressure, µ is viscosity, and g is the gravity vector.

[0747] This model can demonstrate the effects of varying scale and stirring rate on velocity and energy dissipation rate (EDR) within a bioreactor. Uniform volume-averaged hydrodynamic parameters are desirable. If the fluid velocity is insufficient for effective mixing, dead zones can form where mixing does not occur. EDR is often used as a substitute for shear stress in flow condition characterization. If the EDR is too low, aggregates may become too large and clump together, eventually settling. If the EDR is too high, aggregates may be sheared apart, leaving single cells that cannot form aggregates. These conditions lead to reduced cell proliferation, increased cell death, and a lack of uniformity in shape and size of the aggregates.

[0748] The use of conventional bioreactors has demonstrated a hydrodynamic environment unfavorable to the maintenance and growth of iPSCs. Conventional bioreactors provide an environment where high shear stress can damage cells by tearing cells and cell aggregates, reducing cell yield and quality. Shear stress increases with scale, and only a moderate fold increase is observed during the expansion phase in conventional bioreactors. Therefore, the use of conventional bioreactors limits the ability to scale up inoculation and harvest phases.

[0749] Computational fluid dynamics models confirm that vertical wheel bioreactors generate more uniform fluid dynamics by combining radial and axial flow components. Vertical wheel bioreactors exhibit bihnocentric velocity streamlines, while conventional bioreactors have circular velocity streamlines. Compared to conventional bioreactors, vertical wheel bioreactors offer reduced dissipated energy gradients, lower shear effects, and more uniform particle suspension, supporting better scale-up of the vertical wheel bioreactor environment compared to conventional bioreactors.

[0750] High cell fold expansion and morphologically healthy and uniform aggregated cell growth have been observed at a scale of 0.1 L with a stirring rate of 60 rpm. Using relevant equations, these values ​​were correlated with 0.5 L (stirring rate 30 rpm), 3 L (stirring rate 24 rpm), and 15 L (stirring rate 15 rpm) scales to maintain the same volume-average EDR during scaling-up. The proposed working range for volume-average EDR in culturing human iPSCs is between 3.0E and 4m. 2 / s 2 and 1.5E-3m 2 / s 2 between.

[0751] In some embodiments, the cell concentration in the first culture medium is from about 80,000 cells / mL to about 180,000 cells / mL in a volume of about 50 mL to about 70 mL. In one embodiment, the cell concentration in the first culture medium is about 150,000 cells / mL in a volume of about 60 mL. In some embodiments, the cell concentration in the first culture medium is from about 80,000 cells / mL to about 120,000 cells / mL in a volume of about 0.09 L to about 0.11 L. In some embodiments, the cell concentration in the first culture medium is about 100,000 cells / mL in a volume of about 0.1 L. In some embodiments, the cell concentration in the first culture medium is from about 5,000 cells / mL to about 180,000 cells / mL in a volume of about 0.45 L to about 0.55 L. In some embodiments, the cell concentration in the first culture medium is about 20,000 cells / mL in a volume of about 0.5 L.

[0752] In some embodiments, the vertical wheel is perpendicular to the bioreactor vessel. In some embodiments, the vertical wheel is tilted at an angle of approximately 5° to approximately 60° relative to the bioreactor vessel. In one embodiment, the vertical wheel is tilted at an angle of approximately 20° relative to the bioreactor vessel. In one embodiment, the vertical wheel is tilted at an angle of approximately 40° relative to the bioreactor vessel.

[0753] In some embodiments, the bioreactor may be a fed-batch bioreactor. In some embodiments, the bioreactor may be perfused or perfusable. Perfusion is a method in which cells are held within the bioreactor while the culture medium is continuously replaced. Fresh culture medium is added to replenish carbon sources and other nutrients. Depleted culture medium and cellular waste are removed. In some embodiments, perfusion is performed via cell binding, wherein cells are held in the bioreactor by binding to a capillary membrane or filament. In some embodiments, the bioreactor contains a capillary membrane or filament. In some embodiments, perfusion is performed via filtration, wherein used culture medium flows through a membrane having pores too small to accommodate cells. In some embodiments, the bioreactor contains a membrane having pores too small to accommodate cells. In some embodiments, perfusion is performed via sedimentation, wherein cells are allowed to settle and the remaining suspension is removed as used culture medium. In some embodiments, cells settle within the bioreactor.

[0754] In some embodiments, the bioreactor may contain multiple bioreactor wheels. In some embodiments, the bioreactor contains 2 to 6 bioreactor wheels. In some embodiments, the bioreactor contains 2, 3, 4, 5, or 6 bioreactor wheels. In some embodiments, one or more bioreactor wheels are perpendicular to the bioreactor vessel. In some embodiments, one or more bioreactor wheels are not perpendicular to the bioreactor vessel. In some embodiments, one or more bioreactor wheels rotate at about 10 rpm to about 60 rpm. In some embodiments, one or more bioreactor wheels rotate at about 60 rpm. In some embodiments, one or more bioreactor wheels rotate at about 30 rpm. In some embodiments, one or more bioreactor wheels rotate at about 24 rpm. In some embodiments, one or more bioreactor wheels rotate at about 15 rpm.

[0755] Example

[0756] The following examples are provided to further describe some embodiments of the implementations disclosed herein. These examples are intended to illustrate, and not limit, the disclosed implementations.

[0757] Example 1: Serum-free, feeder-free, three-dimensional redifferentiation of γδ T cell-derived iPSCs into γδ T cells .

[0758] The provided examples contain instructions for maintaining and redifferentiating γδ T-iPSCs.

[0759] Material

[0760] The following abbreviations are used in this article (Table 1).

[0761] Table 1. Abbreviations .

[0762]

[0763] The materials required for this study include the γδ T-iPSC line and the reagents necessary for cell culture (Tables 2 to 6).

[0764] Table 2. Reagents used for iPSC culture and maintenance .

[0765]

[0766] Table 3. Reagents required for hematopoietic redifferentiation (listed by the date of redifferentiation) .

[0767]

[0768] Table 4. Reagents used for iγδT cell redifferentiation .

[0769]

[0770] Table 5. Supplies and consumables required for cell culture .

[0771]

[0772] Table 6. Antibodies in the flow group .

[0773]

[0774]

[0775] Table 7. Equipment .

[0776]

[0777] Programs for maintaining γδ T cell-derived induced pluripotent stem cell (γδ T-iPSC) lines .

[0778] 1.1 Prepare supplemented StemFit as follows ® Basic 04 Complete Culture Medium:

[0779] a. StemFit is provided in frozen form. ® Basic 04 complete culture medium should be stored at -20°C or below until used. Supplemented StemFit culture medium should be prepared using aseptic techniques. ® Complete culture medium.

[0780] b. Place the frozen StemFit at room temperature (15°C-25°C) or in a refrigerator (2°C-8°C) with occasional mixing. ®Thaw the Basic 04 complete culture medium.

[0781] c. Note: Do not thaw the culture medium at 37°C, as this will accelerate the degradation of the medium components.

[0782] d. Supplement StemFit with an additional 20 ng / mL of bFGF ® Basic 04 complete medium, to achieve a final concentration of 100 ng / mL bFGF in supplemented medium.

[0783] e. Store the supplemented culture medium at 4°C for up to two (2) weeks.

[0784] f. Allow the culture medium to reach room temperature (15°C-25°C) for at least one (1) hour before use.

[0785] 1.2 Prepare a 10 mM Y-27632 ROCK inhibitor stock solution as follows:

[0786] aY-27632 ROCK inhibitor is received as a crystalline solid.

[0787] b. Store the inhibitor at -20°C for up to 12 months from the date of receipt.

[0788] c. Prepare a 10 mM stock solution in 1X DPBS.

[0789] i. Add 312 μL of 1X DPS to 1 mg Y-27632 and completely resuspend the solution.

[0790] ii. Aliquot 25 μL of the stock solution into Ebendorf tubes labeled Y-27632.

[0791] iii. The aliquots are stable at -20°C for up to 6 months.

[0792] iv. Divide the working volume into equal parts to avoid repeated freeze-thaw cycles.

[0793] 1.3 A 6-well TC processing board coated with iMatrix 511 is prepared as follows:

[0794] a. Transfer 12 mL of 1X DPBS into a 15 mL conical tube.

[0795] b. Add 30 μL of iMatrix 511 to 1X DPBS and mix thoroughly immediately (coating concentration: 0.25 μg / cm). 2 ).

[0796] c. Add 2 mL of iMatrix solution to each well of the 6-well plate.

[0797] d. Incubate the plate at 37°C and 5% CO2 for at least 60 minutes (the plate can be left at 37°C overnight).

[0798] e. Note: After the coating step, the plate can be stored at 4°C for one week.

[0799] 1.4 StemFit supplemented with 10 μM Y-27632 ROCK inhibitor was prepared as follows. ® Basic 04 complete culture medium (from step 1.1):

[0800] a. Thaw the aliquots of the 10mM Y-27632 ROCK inhibitor before use.

[0801] b. Add 25 mL of the supplemented StemFit ® Divide the Basic 04 complete culture medium (from step 1.1) into 50 mL conical tubes.

[0802] c. Add 25 μL of 10 mM Y-27632 ROCK inhibitor to the supplemented StemFit ® In Basic 04 complete medium, to achieve a final concentration of 10 μM Y-27632 ROCK inhibitor.

[0803] 1.5 Thaw γδ T-iPSC vials as follows:

[0804] a. Thaw a small vial of frozen γδ T-iPSCs in a 37°C water bath for 1 minute or until small pieces of frozen floating cells are visible.

[0805] b. Slowly transfer the thawed γδ T-iPSC dropwise from the vial to a 15 mL conical tube containing 9 mL of StemFit supplemented with a 10 µm Y-27632 ROCK inhibitor at room temperature. ® 04 Basic complete culture medium (from step 1.4).

[0806] c. Note: The total volume is 10 mL.

[0807] 1.6 Cell counts were measured as follows:

[0808] a. Use the NucleoCounter NC-200 instrument for cell counting purposes.

[0809] b. Take 150 μL of the sample from the cell suspension (step 1.5b) into a 1.5 mL Ebendorf tube.

[0810] c. Collect cells from the Eppendorf tube into the Via1-detection kit.

[0811] d. Insert the Via1-detection kit into the NC-200 cell counter and count the cells.

[0812] e. Note: Only the total number of live cells should be used for cell seeding purposes.

[0813] 1.7 The following steps were performed for washing and plating of thawed γδ T-iPSCs:

[0814] a. Centrifuge a 15 mL conical tube containing the thawed γδ T-iPSC (step 1.5 b) at 300 × g for 3 minutes at room temperature.

[0815] b. Aspirate the supernatant without disturbing the cell pellet by leaving 0.5 mL of culture medium.

[0816] c. Gently tap the conical tube to loosen the sediment.

[0817] d. Add 1 mL of StemFit supplemented with 10 μM Y-27632 ROCK inhibitor. ® Add P1000 to Basic 04 complete culture medium (step 1.4).

[0818] e. Add supplemental StemFit with 10 μM Y-27632 ROCK inhibitor. ® Basic 04 complete medium (step 1.4) to achieve 1×10 4 The final cell concentration is cells / mL.

[0819] f. Remove iMatrix 511 solution from the 6-well plate by aspiration.

[0820] i. Note: Avoid allowing the plate to dry for an extended period (e.g., more than 10 minutes). Drying of the holes is not permitted.

[0821] g. Mix the cells thoroughly and then plate them at 2 mL / well.

[0822] h. Distribute the cells evenly at the bottom of the well by gently shaking the plate back and forth and left and right manually.

[0823] i. Incubate the plate overnight at 37°C with 5% CO2.

[0824] Day 1 of 1.8: Change the culture medium as follows:

[0825] a. To supplement StemFit ®Basic 04 complete culture medium (from step 1.1) was brought to room temperature from 4°C.

[0826] b. Gently aspirate the culture medium from the 6-well plate.

[0827] c. Add 2 mL / well of freshly replenished StemFit ® Basic 04 complete medium (without ROCK inhibitor) was added to a 6-well plate.

[0828] d. Image the board using EVOS M7000 and record images at 4X and 10X magnification.

[0829] e. Take three images of different regions on the plate to record the morphology and landscape of the culture. Single-cell and two-cell colonies are present. Due to the presence of ROCK inhibitors, the cells exhibit a spiky morphology.

[0830] i. Note: As cells divide, the boundaries become more defined.

[0831] ii. At 10X magnification, the cells show more defined boundaries (data not shown).

[0832] 1.9 Continue culturing cells until they reach their typical morphology, exhibiting all iPSC characteristics, including round colonies with densely packed cells, a high nucleus-to-cytoplasm ratio, well-defined edges, and clear boundaries. At this stage, the iPSC colony confluence is approximately 70%-80%.

[0833] a. Note: It may take 7 days + / - 1 day for a settlement to show this form.

[0834] b. iPSC colonies have defined boundaries and dense cells.

[0835] i. At 10X magnification, iPSC colonies appear to have well-defined boundaries and dense cells (data not shown).

[0836] 1.10 Passage the cells and confirm pluripotency by flow cytometry before initiation of redifferentiation:

[0837] a. As in step 1.3, prepare as many iMatrix 511-coated 6-well plates as needed to pass on the iPSCs.

[0838] b. Perform iPSC transfer as follows:

[0839] i. Aspirate the culture medium from the cells plated in step 1.7.

[0840] ii. Rinse the well with 1 mL of 1X DPBS.

[0841] iii. Draw 1X DPBS out of the well.

[0842] iv. Add 1 mL TrypLE.

[0843] v. Incubate the plate at 37°C for 6 to 8 minutes.

[0844] vi. To examine the wells under a microscope. The cells appear bright with clearly defined cell boundaries (data not shown).

[0845] vii. Gently remove TrypLE.

[0846] viii. Add 1 mL of supplemented StemFit containing 10 μM Y-27632 ROCK inhibitor. ® Add P1000 to Basic 04 complete medium (from step 1.4), and then gently pipette to remove the cells from the plate. Avoid forming air bubbles.

[0847] c. Examine the wells with a microscope to ensure that 95% of the cells have been removed from the wells.

[0848] i. If residual cells still adhere to the plate, add 1 mL of StemFit containing 10 μM Y-27632 ROCK inhibitor. ® Basic 04 complete culture medium (from step 1.4). Scrape the wells with a cell scraper and transfer the solution three (3) times with P1000 to ensure a homogeneous single-cell suspension.

[0849] d. Collect the contents of the well into a 15 mL conical tube.

[0850] e. Perform cell counting as in step 1.6.

[0851] f. 1×10 6 One cell was used for pluripotency flow cytometry analysis.

[0852] g. The multipotency flow groups are shown in Table 6.

[0853] h. Meeting the acceptance criteria prior to the redifferentiation step. An example acceptance criterion for pluripotency is:

[0854] >85% Oct4+, Sox2+, SSEA3+, SSEA4+

[0855] <1% SSEA1+

[0856] <5% CD34+

[0857] A procedure for redifferentiating γδ T-iPSC lineage into hematopoietic stem cells (HSCs) using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method.

[0858] 1.11 Day 0: Hematopoietic redifferentiation begins—mesodermal induction period.

[0859] a. StemFit ® Basic 04 complete culture medium (step 1.1) or StemFit ® Basic 03 medium was preheated at room temperature, and the culture medium from step 1 (Table 8) was prepared for use from day 0 to day 2.

[0860] Table 8. Culture medium for step 1

[0861]

[0862] b. Record the image on day 0.

[0863] c. Perform culture medium exchange. With the brake set to position 6, rotate the entire cell suspension at 100g for 1 minute. Remove the reagents to thaw. Take 500 μL of metabolic sample and remove the remaining supernatant. Resuspend the cells in fresh culture medium from step 1 (Table 8) and add it to the vertical wheel bioreactor. Then add growth factors to the reactor.

[0864] d. Culture the cells at 37°C and 5% CO2.

[0865] Day 2 of 1.12: Hematopoietic redifferentiation begins—from mesoderm to specific hematopoietic lineage specialization.

[0866] a. On day 2, preheat the advanced DMEM / F12 medium at room temperature and prepare the step 2 medium (Table 9) or alternative step 2 medium (Table 10) for use from day 2 to day 4.

[0867] Table 9. Culture medium for step 2

[0868]

[0869] Table 10. Alternative culture media for step 2

[0870]

[0871] b. Record the image on the second day as in step 1.11b.

[0872] c. Perform culture medium exchange. Allow the aggregates to settle in the reactor for approximately 5 minutes. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. Rotate the cell suspension at 100g for 1 minute at brake position 6. Remove the reagents to thaw. Take 500 μL of metabolic sample and remove the remaining supernatant. Resuspend the cells in fresh Step 2 medium (Table 9) or fresh alternative Step 2 medium (Table 10) and add it back to the reactor. Then add growth factors to the reactor.

[0873] d. Incubate and culture the cells at 37°C and 5% CO2 for two days.

[0874] Day 4, January 13: Hematopoietic redifferentiation begins—angiogenic cell induction period.

[0875] a. On day 4, preheat the advanced DMEM / F12 medium at room temperature and prepare the step 3 medium (Table 11) or alternative step 3 medium (Table 12) for use from day 4 to day 6.

[0876] Table 11. Culture medium for step 3

[0877]

[0878] Table 12. Alternative culture media for step 3

[0879]

[0880] b. Record the image on day 4 as in step 1.11b.

[0881] c. Perform culture medium exchange. Allow the aggregates to settle in the reactor for approximately 5 minutes. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. Rotate the supernatant at 100g for 1 minute at brake position 6. Remove the reagents to thaw. Take 500 μL of metabolic sample and discard the remaining supernatant. Resuspend the cells in fresh Step 3 medium (Table 11) or an alternative Step 3 medium (Table 12). Add the solution back to the reactor. Then add the growth factors to the reactor.

[0882] d. Incubate and culture the cells at 37°C and 5% CO2 for two days.

[0883] Day 6, January 14: Hematopoietic redifferentiation begins—the transitional stage from endothelial cells to hematopoietic cells.

[0884] a. On days 6 and 8, preheat the advanced DMEM / F12 medium at room temperature and prepare fresh step 4 medium (Table 13) or alternative step 4 medium (Table 14).

[0885] Table 13. Culture medium for step 4

[0886]

[0887] Table 14. Alternative culture media for step 4

[0888]

[0889] b. Record the image on day 6 as in step 1.11b.

[0890] c. Perform culture medium exchange. Allow the aggregates to settle in the reactor for approximately 5 minutes. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. Rotate the supernatant at 100g for 1 minute at brake position 6. Remove the reagents to thaw. Take 500 μL of metabolic sample and discard the remaining supernatant. Resuspend the cells in fresh Step 4 medium (Table 13) or an alternative Step 4 medium (Table 14). Add the solution back to the reactor. Then add the growth factors to the reactor.

[0891] d. Incubate and culture the cells at 37°C and 5% CO2 for two days.

[0892] e. On day 8, prepare 48 mL of the step 4 medium (Table 13) or an alternative to the step 4 medium (Table 14).

[0893] f. Allow the aggregates to settle in the reactor for approximately 5 minutes. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. Rotate the supernatant at 300g for 5 minutes. Remove the reagents to thaw. Take 500 μL of metabolic sample and discard the remaining supernatant. Resuspend the cells in fresh Step 4 medium (Table 13) or an alternative Step 4 medium (Table 14). Add the solution back to the reactor. Then add the growth factors to the reactor.

[0894] g. Incubate and culture the cells at 37°C and 5% CO2 for another two days.

[0895] Day 10, January 15th: Harvesting hematopoietic stem cells (iHSC / iHSC) and preparing cells for flow cytometry analysis and cryopreservation, as follows:

[0896] a. Record the image on day 10 as in step 1.11b.

[0897] b. Prepare 21 mL of the basal culture medium from step 4 (step 1.14a) which does not contain cytokines and growth factors.

[0898] c. Process single-cell fractionation as follows:

[0899] i. Remove the entire cell volume from the reactor and transfer 30 mL to each of the two 50 mL Erlenmeyer flasks, rotating at 100 g for 1 minute at brake position 6. The pellet will contain clusters, and single cells will remain in the supernatant. Discard the supernatant and resuspend the cells in 2 mL of collagenase II. Incubate the cells at 37°C for 20 minutes.

[0900] ii. Transfer the supernatant containing the single cells to a new 50 mL Erlenmeyer flask. Rotate at 300 g for 5 minutes in brake position 9.

[0901] iii. After centrifugation, collect 500 μL of the cell supernatant for metabolic samples. Remove the remaining supernatant and resuspend the precipitate in 5 mL of the basal medium from step 4 (step 1.15b) without the addition of cytokines or growth factors.

[0902] iv. Cell counting was performed using an NC-200.

[0903] d. Process cluster cell fractionation as follows:

[0904] i. Add 1 mL of collagenase to each precipitate from step 1.15ci. Resuspend and combine in a 50 mL Erlenmeyer flask (total volume of collagenase = 2 mL). Incubate at 37°C for 20 minutes.

[0905] ii. Add an equal volume (2 mL) of the basal culture medium from step 4 (step 1.15.b) without added cytokines or growth factors. Centrifuge at 300 g for 5 minutes at brake setting 9.

[0906] iii. Remove the supernatant, add 2 mL TrypLE, and mix the solution. Incubate at 37°C for 5 minutes.

[0907] iv. Add 8 mL of the basal medium from step 4 (step 1.15.b) without added cytokines or growth factors and mix the solution.

[0908] v. Pass the cells through a 70µm filter and centrifuge at 300g for 5 minutes at brake setting 9.

[0909] vi. Resuspend in 10 mL of the basal medium from step 4 (step 1.15.b) without the addition of cytokines or growth factors, and perform cell counting using an NC-200.

[0910] e. 2.0 × 10 6 Each cell was aliquoted and used for flow cytometry analysis using the iHSC group.

[0911] The f.iHSC flow group is shown in Table 6.

[0912] Exemplary success criteria for the g.iHSC redifferentiation step: purity of each input iPSC >70% lineage-CD34+ cells and fold expansion >5.

[0913] This approach enables the γδ T-iPSC line to redifferentiate into hematopoietic stem cells (HSCs) using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method in a 0.1L vertical wheel bioreactor with multiple RPMs, resulting in improvements in cell cluster formation, morphology, cell growth (e.g., metabolism), and purity. The same applies to perfusion within the bioreactor system.

[0914] Example 2: Serum-free, feeder-free redifferentiation of γδ T cell-derived iPSCs into γδ T cells .

[0915] The provided examples include instructions for redifferentiating γδ T-iPSCs using GMP-compliant reagents or GMP-like reagents.

[0916] Material

[0917] The following abbreviations were used in this article (Table 15).

[0918] Table 15. Abbreviations .

[0919]

[0920] The materials required for this study include the γδ T-iPSC line and the reagents necessary for each cell culture step (Tables 16 to 21).

[0921] Table 16. Reagents used for iPSC culture and maintenance .

[0922]

[0923] Table 17. Reagents required for iCD34 redifferentiation (listed by redifferentiation date)

[0924]

[0925]

[0926] Table 18. Supplies and Consumables Required for Cell Culture .

[0927]

[0928] Table 19. Reagents used in flow cytometry .

[0929]

[0930] Table 20. Antibodies in pluripotent mobile groups .

[0931]

[0932] Table 21. Antibodies in the iCD34 mobile group .

[0933]

[0934] Table 22. Software .

[0935]

[0936] Table 23. Equipment .

[0937]

[0938] Table 24. BD LSRFortessa ™ Cell instrument configuration .

[0939]

[0940] Table 25. FACSymphony ™ Configuration of A5 cell analyzer .

[0941]

[0942] Procedures for maintaining iPSC .

[0943] 2.1 Prepare supplemented StemFit as follows ® Basic 04 Complete Culture Medium:

[0944] a. StemFit is provided in frozen form. ® Basic 04 complete culture medium should be stored at -20°C or below until used. Supplemented StemFit culture medium should be prepared using aseptic techniques. ® Complete culture medium.

[0945] b. Place the frozen StemFit at room temperature (RT; 15℃-25℃) or in a cold storage (2℃-8℃) with occasional mixing. ® Thaw the Basic 04 complete culture medium overnight.

[0946] c. Note: Do not thaw the culture medium at 37°C, as this will accelerate the degradation of the medium components.

[0947] d. Supplement StemFit with an additional 20 ng / mL of bFGF ® Basic 04 complete medium, to achieve a final concentration of 100 ng / mL bFGF in supplemented medium.

[0948] e. Store the supplemented culture medium at 4°C for up to two (2) weeks.

[0949] f. Allow the culture medium to reach RT at least one (1) hour before use.

[0950] 2.2 Prepare a 10 mM Y-27632 ROCK inhibitor stock solution as follows:

[0951] aY-27632 ROCK inhibitor (ROCKi) is received as a crystalline solid.

[0952] b. Store the inhibitor at -20°C for up to 12 months from the date of receipt.

[0953] c. Prepare a 10 mM stock solution in 1X DPBS.

[0954] i. Add 312 μL of 1X DPS to 1 mg Y-27632 and completely resuspend the solution.

[0955] ii. Aliquot 25 μL of the stock solution into Ebendorf tubes labeled Y-27632.

[0956] iii. The aliquots are stable at -20°C for up to 6 months.

[0957] iv. Divide the working volume into equal parts to avoid repeated freeze-thaw cycles.

[0958] 2.3 The iPSCs were directly thawed into 3D cultures as follows:

[0959] a. Obtain a ROCKi aliquot from -20°C and thaw to room temperature.

[0960] b. Warm 500 mL of StemFit Basic 04 complete medium to 37°C.

[0961] c. For a 0.5L vertical wheel platform, transfer 490mL of complete culture medium and 500μL of ROCKi into a dish and place it on the magnetic base in the incubator while stirring at 30rpm. Let the dish stand in the incubator for at least 1 hour before adding cells.

[0962] d. Warm 25 mL of fresh culture medium in a water bath. This culture medium does not contain ROCKi.

[0963] e. Remove the frozen iPSC vial and thaw it in a 37°C water bath.

[0964] f. Transfer the iPSC cell solution to a 15 mL tube. Wash the frozen tube with 1 mL of warm culture medium and add it to the 15 mL tube. Fill the 15 mL tube with the thawed cell solution to a total of 10 mL and mix gently.

[0965] g. Centrifuge iPSC cells at 300g for 5 minutes at room temperature. Remove the supernatant and resuspend the cells in 5 mL of warm culture medium. Collect 2 × 200 μL of sample into Eppendorf tubes and count the cells on an NC200.

[0966] h. Using NC200 counting, calculate the cell volume required to achieve a seeding density of 20,000 cells / mL.

[0967] i. Collect cells from the Eppendorf tube into the Via1-detection kit.

[0968] ii. Insert the Via1-detection kit into the NC-200 cell counter and count the cells.

[0969] iii. Note: Only the total number of live cells should be used for cell seeding purposes.

[0970] i. Transfer the calculated cell volume to the preheated culture medium in the dish. Fill the dish to a total cell volume of 500 mL and place it back on the base of the incubator to incubate at 37°C for three days.

[0971] 2.4 The cell culture medium for 3D cultures is exchanged as follows:

[0972] a. Perform a 50% culture medium replacement on days 4, 5, and 6. The culture medium replacement should not contain ROCKI.

[0973] b. Prepare equal aliquots to replace half of the total culture medium volume and place them in a water bath at 37°C for ≥30 minutes.

[0974] c. Remove the dish from the incubator and place it in a fume hood to allow the cells to settle inside the dish.

[0975] d. The vessel remains undisturbed during the cell settling time.

[0976] e. The aggregates did not settle excessively. Large aggregates took about 5 minutes to settle, while smaller aggregates took about 10 minutes to settle.

[0977] f. While the wheel remains stationary, remove 50% of the culture medium from the vessel and discard the supernatant.

[0978] g. Add fresh culture medium to replace the volume removed from the vessel.

[0979] h. Place the reactor back on the base in the 37°C incubator and repeat the cell culture medium replacement process daily until harvest on the 7th day.

[0980] 2.5 The following procedure is followed to harvest iPSCs from VW cultures:

[0981] a. On day 7 of iPSC 3D culture, divide 100 mL of TrypLE into equal portions, and add 1 μL of ROCKi to each 1 mL of TrypLE aliquot to produce a concentration of 10 μM.

[0982] b. Remove the vessel from its base and place it in the BSC to allow the aggregate to settle for 3 to 5 minutes (3 minutes for larger aggregates).

[0983] c. The vessel remains undisturbed during the settling period.

[0984] d. After sedimentation, carefully remove the used culture medium from the dish into a container using a serum pipette until less than 10 mL of used culture medium remains in the dish.

[0985] i. First, use a 50 mL or 25 mL serum pipette to remove most of the used culture medium.

[0986] ii. Next, use a 10 mL serum pipette to remove the remaining culture medium, leaving <10 mL (culture medium and cells) in the dish.

[0987] e. Discard the supernatant and add 100 mL TrypLE+ROCKi to the dish to dissociate the cells within the dish.

[0988] f. Mount the vessel onto its base inside the incubator and stir at 50 rpm for 20 minutes to promote enzymatic cell dissociation.

[0989] g. Once dissociation is complete, remove the vessel and transfer 25 mL of the solution into four 50 mL centrifuge tubes, each containing approximately 25 mL of cell solution. If any extra volume remains in the reactor, distribute it across the conical tubes. Mix the solution in each conical tube using a 10 mL pipette until no clusters are visible (approximately 5 to 10 mixes). Once complete dissociation is confirmed, add PBS buffer until a total volume of 50 mL is reached. Then mix the solutions.

[0990] h. Centrifuge the cells at 300g for 5 minutes.

[0991] i. Once centrifugation is complete, discard the supernatant using a serum pipette (or aspirator).

[0992] j. Using a micropipette, add 2 mL of fresh culture medium to each tube and gently resuspend the cell pellet. Combine the cell solutions in a conical tube to reach a total of 8 mL of cell solution.

[0993] k. Take a 150 μL sample from the vessel and add it to a microtube for counting.

[0994] 1. Add 150 μL of PBS to the microtube containing the cell sample, diluting it 2×.

[0995] m. Cell count was performed using an NC-200. Live cells / mL, cell viability, cell diameter, and percentage of remaining aggregates were recorded.

[0996] The next step is to proceed to step 2.6 for iCD34 differentiation, seeding cells at 20,000 cells / mL into a new 0.5L dish for further iPSC amplification, or freeze the sample for flow cytometry analysis by centrifuging the required number of cells at 300g for 5 minutes, removing the supernatant, resuspending the cells in 1 mL CS10, and then storing them overnight at -80°C.

[0997] i. Divide 5×10 6 One cell was used for pluripotency flow cytometry analysis.

[0998] ii. The pluripotency flow groups are shown in Table 20.

[0999] iii. It meets the acceptance criteria before undergoing the redifferentiation step. An example acceptance criterion for pluripotency is:

[1000] >85% Oct4+, Sox2+, SSEA3+, SSEA4+

[1001] <1% SSEA1+

[1002] <5% CD34+

[1003] Using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method in a vertical wheel bioreactor to cultivate γδ The program for T-iPSC clones to redifferentiate into iPSC-derived CD34 (iCD34) cells .

[1004] 2.6 Day 0: Hematopoietic redifferentiation begins—mesodermal induction period.

[1005] a. StemFit ® The Basic 04 complete medium (step 2.1) was preheated at RT and the step 1 medium (Table 26) was prepared for day 0 to day 2.

[1006] Table 26. Culture medium used for Step 1 from Day 0 to Day 2 .

[1007]

[1008] b. Harvest cells from the plate and obtain cell counts as in step 2.10be.

[1009] c. Add 60 mL of the culture medium from step 1 to the reactor at a seeding density of 150,000 cells / mL.

[1010] d. The cells were cultured at 37°C and 5% CO2, with the reactor wheel rotating at 40 rpm.

[1011] 2.7 Day 2: Hematopoietic redifferentiation begins—from mesoderm to specific hematopoietic lineage specialization.

[1012] a. On day 2, preheat the advanced DMEM / F12 medium at RT.

[1013] b. On the second day, record the images.

[1014] i. Transfer the reactor to the spare reactor base within the BSC.

[1015] ii. When the reactor wheel is rotating at 40 rpm, remove 4 mL of cell suspension and transfer it to one well of an ultra-low adhesion 6-well plate.

[1016] iii. Gently vortex the plate to concentrate the cells in the center of the well, and then image the cells.

[1017] iv. Once the images have been recorded, transfer the cells back to the reactor. Vortex the plate to concentrate the cells in the center of the wells, remove 1-2 mL from the center of the wells, and then tilt the plate forward and backward to remove cells from the top of the wells. Starting from the top of the culture medium level, tilt the plate forward and aspirate the remaining cells by slowly moving the pipette back and forth. Transfer the cells to the reactor.

[1018] c. Remove the reactor from the base and allow the aggregates to settle in the reactor for approximately 5 minutes or longer, until the clusters in the suspension are no longer visible and have moved to the bottom of the reactor. After about 2 minutes, gently shake the 0.1L reactor a few times to ensure that the clusters settled at the top of the vertical wheel are removed. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. At brake position 6, rotate the removed supernatant at 100g for 1 minute.

[1019] d. While the cells are in the centrifuge, add 38.4 mL of DMEM / F12 and 8.6 mL of StemFit differentiation medium to the empty reactor. Place the reactor back on the base and rotate it at 40 rpm.

[1020] e. Thaw the reagents listed below for the culture medium used in step 2 from day 2 to day 4.

[1021] Table 27. Culture medium used for step 2 from day 2 to day 4 .

[1022]

[1023] f. After centrifugation, collect 500 μL of the cell supernatant for metabolic samples. Remove the remaining supernatant.

[1024] g. Use 1 mL StemFit to remove the precipitate. ® The differentiation medium was resuspended and added to the reactor.

[1025] h. Add the other reagents from step 2 to the reactor.

[1026] i. Incubate and culture the cells at 37°C, 5% CO2, and 40 rpm for two days.

[1027] 2.8 Day 4: Hematopoietic redifferentiation begins—angiogenic cell induction period.

[1028] a. On day 4, preheat the advanced DMEM / F12 medium at RT.

[1029] b. Record the image for day 4 as in step 2.7b.

[1030] c. Remove the reactor from the base and allow the aggregates to settle in the reactor for approximately 5 minutes or longer, until the clusters in the suspension are no longer visible and have moved to the bottom of the reactor. After about 2 minutes, gently shake the 0.1L reactor a few times to ensure that the clusters settled at the top of the vertical wheel are removed. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. At brake position 6, rotate the removed supernatant at 100g for 1 minute.

[1031] d. While the cells are in the centrifuge, add 38.4 mL of DMEM / F12 + 8.6 mL of StemFit differentiation medium to the empty reactor. Place the reactor back on the base and rotate it at 40 rpm.

[1032] e. Thaw the reagents used in step 3 for days 4 through 6.

[1033] Table 28. Culture medium used for step 3 from day 4 to day 6 .

[1034]

[1035] f. After centrifugation, collect 500 μL of the cell supernatant for metabolic samples. Remove the remaining supernatant.

[1036] g. Use 1 mL StemFit to remove the precipitate. ® The differentiation medium was resuspended and added to the reactor.

[1037] h. Add the other reagents from step 3 to the reactor.

[1038] i. Incubate and culture the cells at 37°C, 5% CO2, and 40 rpm for two days.

[1039] 2.9 Day 6: Hematopoietic redifferentiation begins—the transitional stage from endothelial cells to hematopoietic cells.

[1040] a. On day 6, preheat the advanced DMEM / F12 medium at RT.

[1041] b. Record the image on day 6 as in step 2.7b.

[1042] c. Remove the reactor from the base and allow the aggregates to settle in the reactor for approximately 5 minutes or longer, until the clusters in the suspension are no longer visible and have moved to the bottom of the reactor. After about 2 minutes, gently shake the 0.1L reactor a few times to ensure that the clusters settled at the top of the vertical wheel are removed. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. At brake position 6, rotate the removed supernatant at 100g for 1 minute.

[1043] d. While the cells are in the centrifuge, add 38.4 mL of DMEM / F12 + 8.6 mL of StemFit differentiation medium to the empty reactor. Place the reactor back on the base and rotate it at 40 rpm.

[1044] e. Thaw the reagents used in step 4 for days 6 through 8.

[1045] Table 29. Culture medium used in step 4 from day 6 to day 10 .

[1046]

[1047] f. After centrifugation, collect 500 μL of the cell supernatant for metabolic samples. Remove the remaining supernatant. Transfer the precipitate to 1 mL StemFit. ® The differentiation medium was resuspended and added to the reactor.

[1048] g. Add the other reagents from step 4 to the reactor.

[1049] h. Incubate and culture the cells at 37°C, 5% CO2, and 40 rpm for two days.

[1050] i. On day 8, preheat the advanced DMEM / F12 medium at RT and record images as in step 2.7b.

[1051] j. Remove the reactor from the base and allow the aggregates to settle in the reactor for approximately 5 minutes or longer, until the clusters in the suspension are no longer visible and have moved to the bottom of the reactor. After approximately 2 minutes, gently shake the 0.1L reactor a few times to ensure that the clusters settled at the top of the vertical wheel are removed. Then, remove the culture medium from the vertical wheel, leaving 20% ​​of the volume in the reactor. At brake position 6, rotate the removed supernatant at 100g for 1 minute.

[1052] k. While the cells are in the centrifuge, add 38.4 mL of DMEM / F12 + 8.6 mL of StemFit differentiation medium to the empty reactor. Place the reactor back on the base and rotate it at 40 rpm.

[1053] 1. After centrifugation, transfer the supernatant containing single cells to a 50 mL conical tube. Resuspend the precipitate from the first conical tube in 500 µL of StemFit differentiation medium and add it to the reactor.

[1054] m. Transfer the supernatant to a second 50 mL conical tube and centrifuge at 300 g for 5 minutes at brake position 9. After centrifugation, collect 500 μL from the cell supernatant for metabolic samples. Remove the remaining supernatant and resuspend the pellet in 500 μL of StemFit differentiation medium and add it to the reactor.

[1055] n. Thaw the reagents used for the culture medium in step 4 for days 8 to 10 (similar to step 2.9e) and add them to the reactor.

[1056] o. Incubate and culture the cells for another two days at 37°C, 5% CO2, and 40 rpm.

[1057] Day 10 (2.10): Hematopoietic stem cells (iCD34 cells) were harvested and prepared for flow cytometry analysis and cryopreservation, as follows:

[1058] a. Record the image on day 10 as in step 2.7b.

[1059] b. Prepare 21 mL of the basal culture medium from step 4, which does not contain cytokines and growth factors.

[1060] c. Process single-cell fractionation as follows:

[1061] i. Remove the entire cell volume from the reactor and transfer 30 mL into each of the 2 × 50 mL Erlenmeyer flasks, rotating at 100 g for 1 minute at brake position 6. Reserve the precipitate for dissociation in step 2.10d, and dissociate the supernatant containing single cells in the following steps.

[1062] ii. Transfer the supernatant containing the single cell to a new 50 mL conical tube. Rotate at 300 g for 5 minutes in brake position 9.

[1063] iii. After centrifugation, collect 500 µL of the cell supernatant for metabolic samples. Remove the remaining supernatant and resuspend the precipitate in 5 mL of fresh, high-quality DMEM / F12 basal medium without added cytokines or growth factors.

[1064] iv. Perform cell counting using NC-200 as in step 2.3h.

[1065] d. Process cluster cell fractionation as follows:

[1066] i. Add 1 mL of collagenase to each precipitate from step 2.10ci. Resuspend and combine in a 50 mL conical tube (total volume of collagenase = 2 mL). Incubate at 37°C for 20 minutes.

[1067] ii. Add an equal volume (2 mL) of high-grade DMEM / F12 basal medium without added cytokines or growth factors (step 2.15b). Centrifuge at 300 g for 5 minutes at brake setting 9.

[1068] iii. Remove the supernatant, add 2 mL TrypLE, and mix the solution. Incubate at 37°C for 5 minutes.

[1069] iv. Add 8 mL of high-quality DMEM / F12 basal medium without added cytokines or growth factors and mix the solution.

[1070] v. If clumping is observed after dissociation, pass the cells through a 70µm filter and centrifuge at 300×g for 5 minutes at brake position 9.

[1071] vi. Resuspend in 10 mL of advanced DMEM / F12 basal medium without added cytokines or growth factors, and perform cell counting using NC-200 as in step 2.7b.

[1072] e. Remove the supernatant by rotating the cell suspension at 300g for 5 minutes and resuspending it in CS10 formulation. Freeze each cell fraction, single cell, and cluster. 2.0 × 10 6 CS10 aliquots of 1 cell / mL were used for flow cytometry analysis and downstream processing.

[1073] The f.iCD34 flow group is shown in Table 21.

[1074] Exemplary success criteria for the g.iCD34 redifferentiation step: purity [defined as lineage marker negative (Lin-)CD34+] > 70%, and fold increase of > 0.5-fold per input iPSC.

[1075] This approach enables the γδ T-iPSC line to redifferentiate into hematopoietic stem cells (HSCs) using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method in a 0.1L vertical wheel bioreactor with multiple RPMs, resulting in improvements in cell cluster formation, morphology, cell growth (e.g., metabolism), and purity. The same applies to perfusion within the bioreactor system.

[1076] Example 3: Serum-free, feed-free conversion of γδ T cell-derived iPSCs to iCD34 at a 3L bioreactor scale Layer, three-dimensional redifferentiation .

[1077] The provided examples include instructions for redifferentiating γδ T-iPSCs.

[1078] Material

[1079] The following abbreviations are used in this article (Table 30).

[1080] Table 30. Abbreviations .

[1081]

[1082] The materials required for this study include the γδ T-iPSC line and the reagents necessary for cell culture (Tables 31 to 34).

[1083] Table 31. Reagents used for iPSC culture and maintenance .

[1084]

[1085] Table 32. Reagents required for hematopoietic redifferentiation (listed by the date of redifferentiation) .

[1086]

[1087] Table 33. Supplies and Consumables Required for Cell Culture .

[1088]

[1089] Table 34. Antibodies in the mobile group .

[1090]

[1091] Table 35. Equipment .

[1092]

[1093] Procedures for maintaining iPSC .

[1094] 3.1 Prepare the supplemented StemFit as follows ® Basic 04 Complete Culture Medium:

[1095] a. StemFit is provided in frozen form. ® Basic 04 complete culture medium should be stored at -20°C or below until used. Supplemented StemFit culture medium should be prepared using aseptic techniques. ® Complete culture medium.

[1096] b. Place the frozen StemFit at room temperature (RT; 15℃-25℃) or in a cold storage (2℃-8℃) with occasional mixing. ® Thaw the Basic 04 complete culture medium.

[1097] c. Note: Do not thaw the culture medium at 37°C, as this will accelerate the degradation of the medium components.

[1098] d. Supplement StemFit with an additional 20 ng / mL of bFGF ® Basic 04 complete medium, to achieve a final concentration of 100 ng / mL bFGF in supplemented medium.

[1099] e. Store the supplemented culture medium at 4°C for up to two (2) weeks.

[1100] f. Allow the culture medium to reach RT at least one (1) hour before use.

[1101] 3.2 Prepare a 10 mM Y-27632 ROCK inhibitor stock solution as follows:

[1102] aY-27632 ROCK inhibitor (ROCKi) is received as a crystalline solid.

[1103] b. Store the inhibitor at -20°C for up to 12 months from the date of receipt.

[1104] c. Prepare a 10 mM stock solution in 1X DPBS.

[1105] i. Add 312 mL of 1X DPS to 10 mg of Y-27632 and completely resuspend the solution.

[1106] ii. Divide 500 μL of the stock solution into equal portions in Eppendorf tubes labeled ROCKi stock solution.

[1107] iii. The aliquots are stable at -20°C for up to 6 months.

[1108] iv. Divide the working volume into equal parts to avoid repeated freeze-thaw cycles.

[1109] 3.3 The iPSCs were directly thawed into 3D cultures as follows:

[1110] a. Obtain a ROCKi aliquot from -20°C and thaw to room temperature.

[1111] b. Warm 500 mL of StemFit Basic 04 complete medium to 37°C.

[1112] c. For a 0.5L vertical wheel platform, transfer 490mL of complete culture medium and 500μL of ROCKi into a dish and place it on the magnetic base in the incubator while stirring at 30rpm. Let the dish stand in the incubator for at least 1 hour before adding cells.

[1113] d. Warm 25 mL of fresh culture medium in a water bath. This culture medium does not contain ROCKi.

[1114] e. Remove the frozen iPSC vial and thaw it in a 37°C water bath.

[1115] f. Transfer the iPSC cell solution to a 15 mL tube. Wash the frozen tube with 1 mL of warm culture medium and add it to the 15 mL tube. Fill the 15 mL tube with the thawed cell solution to a total of 10 mL and mix gently.

[1116] g. Centrifuge iPSC cells at 300g for 5 minutes at room temperature. Remove the supernatant and resuspend the cells in 5 mL of warm culture medium. Collect 2 × 200 μL of sample into Eppendorf tubes and count the cells on an NC200.

[1117] h. Using NC200 counting, calculate the cell volume required to achieve a seeding density of 20,000 cells / mL.

[1118] i. Collect cells from the Eppendorf tube into the Via1-detection kit.

[1119] ii. Insert the Via1-detection kit into the NC-200 cell counter and count the cells.

[1120] iii. Note: Only the total number of live cells should be used for cell seeding purposes.

[1121] i. Transfer the calculated cell volume to the preheated culture medium in the dish. Fill the dish to a total cell volume of 500 mL and place it back on the base of the incubator to incubate at 37°C for three days.

[1122] 3.4 The cell culture medium for 3D cultures is exchanged as follows:

[1123] a. Perform a 50% culture medium replacement on days 4, 5, and 6. The culture medium replacement should not contain ROCKI.

[1124] b. Prepare equal aliquots to replace half of the total culture medium volume and place them in a water bath at 37°C for ≥30 minutes.

[1125] c. Remove the dish from the incubator and place it in a fume hood to allow the cells to settle inside the dish.

[1126] d. The vessel should remain undisturbed during the cell settling time. After approximately 2 minutes, gently shake the reactor a few times to ensure that any clusters settling at the top of the vertical wheel are removed.

[1127] e. The aggregates did not settle excessively. Large aggregates took about 5 minutes to settle, while smaller aggregates took about 10 minutes to settle.

[1128] f. While the wheel remains stationary, remove 50% of the culture medium from the vessel and discard the supernatant.

[1129] g. Add fresh culture medium to replace the volume removed from the vessel.

[1130] h. Place the reactor back on the base in the 37°C incubator and repeat the cell culture medium replacement process daily until harvest on the 7th day.

[1131] 3.5 The following procedure is followed to harvest iPSCs from VW cultures:

[1132] a. On day 7 of iPSC 3D culture, divide 100 mL of TrypLE into equal portions, and add 1 μL of ROCKi to each 1 mL of TrypLE aliquot to produce a concentration of 10 μM.

[1133] b. Remove the vessel from its base and place it in the BSC to allow the aggregate to settle for 3 to 5 minutes (3 minutes for larger aggregates).

[1134] c. The vessel remains undisturbed during the settling period.

[1135] d. After sedimentation, carefully remove the used culture medium from the dish into a container using a serum pipette until less than 10 mL of used culture medium remains in the dish.

[1136] i. First, use a 50 mL or 25 mL serum pipette to remove most of the used culture medium.

[1137] ii. Next, use a 10 mL serum pipette to remove the remaining culture medium, leaving <10 mL (culture medium and cells) in the dish.

[1138] e. Discard the supernatant and add 100 mL TrypLE+ROCKi to the dish to dissociate the cells within the dish.

[1139] f. Mount the vessel onto its base inside the incubator and stir at 50 rpm for 20 minutes to promote enzymatic cell dissociation.

[1140] g. Once dissociation is complete, remove the container and transfer 25 mL of the solution into four 50 mL centrifuge tubes, each containing approximately 25 mL of cell solution. If any extra volume remains in the reactor, distribute it across the conical tubes. Mix the solution in each conical tube using a 10 mL pipette until no clusters are visible (approximately 5 to 10 mixes). Once complete dissociation is confirmed, add PBS buffer until a total volume of 50 mL is reached. Then mix the solution 2–3 times.

[1141] h. Centrifuge the cells at 300g for 5 minutes.

[1142] i. Once centrifugation is complete, discard the supernatant using a serum pipette (or aspirator).

[1143] j. Using a micropipette, add 2 mL of fresh culture medium to each tube and gently resuspend the cell pellet. Combine the cell solutions in a conical tube to reach a total of 8 mL of cell solution.

[1144] k. Filter the cells into a new conical tube using a 70µm filter.

[1145] 1. Take a 150 μL sample from the conical tube and add it to the microtube for counting.

[1146] m. Add 150 μL of PBS to the microtube containing the cell sample, diluting it 2×.

[1147] n. Use an NC-200 to count the number of cells. Record the percentage of live cells / mL, cell viability, cell diameter, and remaining aggregates.

[1148] o. The next step is to proceed to step 3.6 to initiate the iCD34 differentiation process in a 3L reactor, seeding cells at 20,000 cells / mL into a new 0.5L dish for further iPSC amplification, or to freeze the sample for flow cytometry analysis by centrifuging the required number of cells at 300g for 5 minutes, removing the supernatant, resuspending the cells in 1 mL CS10, and then storing them overnight at -80°C.

[1149] i. Divide 5×10 6 One cell was used for pluripotency flow cytometry analysis.

[1150] ii. Multipotent flow groups are shown in Table 34.

[1151] iii. It meets the acceptance criteria before undergoing the redifferentiation step. An example acceptance criterion for pluripotency is:

[1152] >85% Oct4+, Sox2+, SSEA3+, SSEA4+

[1153] <1% SSEA1+

[1154] <5% CD34+

[1155] Using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method in a 3L vertical wheel bioreactor to cultivate γδ The procedure for T-iPSC lineages to redifferentiate into hematopoietic stem cells (HSCs) .

[1156] 3.6 Day -3 Setup of the PBS 3L reactor (maintain sterility).

[1157] a. Remove the PBS 3MAG container from the packaging three days before vaccination.

[1158] b. Introduce PBS 3MAG into the BSC and carefully remove the shrink-wrapped plastic around it.

[1159] c. Place the PBS 3MAG container upright in the BSC and clamp all tubing.

[1160] d. Introduce the autoclaved bag of the extraction tube into the BSC.

[1161] e. Carefully unscrew the unused port cap (right front port) and place it to the side. Remove the 4µm pipette from the autoclave bag and carefully place it into the glass dish, avoiding contact with the outside. Lower the pipette until the glass is halfway between the wheel and the bottom of the dish. Thread the pipette into the port.

[1162] f. Carefully unscrew the pH probe port (right rear port) and place it to the side. Remove the 40µm pipette from the autoclave bag and carefully place it into the glass dish, avoiding contact with the outside. Lower the pipette until the glass stock is above the bridge of the retaining wheel, with the wheel at 3 / 4 of the height from the bottom of the dish. Thread the pipette into the port.

[1163] g. Transfer the vessel out of the BSC and move it toward the PBS 3MAG control tower.

[1164] h. Insert the PBS 3MAG container into the front holder and carefully ensure that the bottom of the container is in contact with the heating pad on the bottom.

[1165] i. Insert the temperature probe into the thermocouple holder.

[1166] j. Connect the dissolved oxygen (DO) cable to the probe.

[1167] k. Attach the main gas and O2 gas lines from the vessel to the 3MAG connector.

[1168] 1. Install the exhaust filter into the filter oven and place the condenser bag appropriately.

[1169] m. Weld 2L bottles to each draw-out line to collect waste.

[1170] n. Weld the 2L DMEM culture medium bottle to the harvest pipeline.

[1171] o. Use a Masterflex pump to discharge or pump in the culture medium.

[1172] p. Once the culture medium from the bag has been completely transferred, clamp the harvest line.

[1173] q. By pumping the culture medium through each draw tube until it reaches the waste bottle, and then pumping the culture medium back into the vessel, the culture medium is passed through the draw tube.

[1174] r. After the culture medium has been completely transferred, set the following parameters and monitor the sterility of the reactor for three days:

[1175] i. Stirring = 23 RPM

[1176] ii. Temperature = 37℃ / 5% CO2

[1177] iii.DO = Close

[1178] iv. Main gas = 0.20 L / min

[1179] v.pH=off

[1180] Sterility was confirmed after 72 hours.

[1181] t. Take out the sample for offline evaluation via Metaflex or Novaflex.

[1182] 3.7 Day 0: Hematopoietic redifferentiation begins—mesodermal induction period.

[1183] a. Transfer 2.4L of StemFit Basic 04 complete medium to a weldable bottle or bag, and then transfer the medium to an empty 3L dish.

[1184] i. Once the culture medium level in the vessel reaches 0.5L, set the temperature to 37°C and the stirring speed to 23 rpm to preheat the culture medium. Additionally, set the gas flow rate to 0.2L / min.

[1185] b. In another weldable bottle or bag, prepare approximately 50 mL of the culture medium from step 1 for day 0 to day 2, wherein the cell seeding density is 150,000 cells / mL of the total working volume of the reactor (i.e., 375 × 10⁻⁶ for a 2500 mL working volume). 6 (cells).

[1186] Table 36. Culture medium used for Step 1 from Day 0 to Day 2 .

[1187]

[1188] c. Transfer approximately 50 mL of cells + the culture medium from step 1 into the reactor.

[1189] d. Prepare a third weldable bottle with 50 mL of StemFit Basic 04 complete medium without cytokines, and transfer the medium to the reactor to wash the harvest line.

[1190] e. Clamp the harvest line and the two extraction lines.

[1191] f. The cells were cultured at 37°C and 5% CO2, with the reactor wheel rotating at 23 rpm.

[1192] 3.8 Day 2: Hematopoietic redifferentiation begins—from mesoderm to specific hematopoietic lineage specialization.

[1193] a. On day 2, preheat the advanced DMEM / F12 medium and StemFit differentiation medium at RT.

[1194] b. On the second day, record the images.

[1195] i. Take 4 mL of cell suspension through the sample line and transfer it to one well of an ultra-low adhesion 6-well plate.

[1196] ii. Gently vortex the plate to concentrate the cells in the center of the well, and then image the cells.

[1197] iii. Once the image is recorded, discard the plate.

[1198] c. Transfer 2720 mL of advanced DMEM / F12 medium + 680 mL of StemFit differentiation medium to a weldable bottle or bag, and connect the bottle or bag to the reactor harvest line.

[1199] i. Alternatively, the volume is divided into a first bottle or bag containing 1600 mL DMEM / F12 medium + 400 mL StemFit differentiation medium (total volume 2L) and a second bottle or bag containing 1120 mL DMEM / F12 medium + 280 mL StemFit differentiation medium (total volume 1.4L).

[1200] d. Weld the empty waste bottle to the 40µm suction tube waste line.

[1201] e. Simultaneously, the culture medium is removed from the reactor through a 40µm pick-up line, and the culture medium is added back to the reactor at a rate of 30mL / min via a harvest line until 3.4L of culture medium is replaced.

[1202] i. Track replacements by ensuring the liquid level indicated on the vessel screen remains consistent. If the liquid level is not consistent, temporarily stop the pump in the harvest or draw line until the liquid level returns to its original value.

[1203] f. Prepare approximately 50 mL of the culture medium for step 2 from day 2 to day 4 in another weldable bottle or bag, and weld the bottle or bag to the harvest line.

[1204] Table 37. Culture medium used for step 2 from day 2 to day 4 .

[1205]

[1206] g. Once 3.4 L of culture medium has been replaced in the vessel, remove 0.1 L of culture medium through the 40 µm pipette. Track the volume removed by monitoring the liquid level on the vessel screen.

[1207] h. Add 50 mL of the culture medium from step 2 to the reactor.

[1208] i. Prepare another weldable bottle or bag using 40 mL of advanced DMEM / F12 medium + 10 mL of StemFit differentiation medium, and weld the bottle or bag to the harvest pipeline.

[1209] j. Transfer 50 mL of DMEM / F12 medium + StemFit differentiation medium into the reactor.

[1210] k. Clamp the harvesting and extraction lines.

[1211] l. Incubate and culture the cells at 37°C, 5% CO2, and 23 rpm for two days.

[1212] 3.9 Day 4: Hematopoietic redifferentiation begins—angiogenic cell induction period.

[1213] a. On day 4, preheat the advanced DMEM / F12 medium and StemFit differentiation medium at RT.

[1214] b. On day 4, record the images as in step 3.8b.

[1215] c. Transfer 2720 mL of advanced DMEM / F12 medium + 680 mL of StemFit differentiation medium to a weldable bottle or bag. Alternatively, aliquot the volume into two bottles or bags as in step 3.8ci. Remove the medium from the reactor through a 40 µm pick-up line as in step 3.8de, and add fresh medium to the reactor via a harvest line.

[1216] d. Prepare 50 mL of the culture medium from day 4 to day 6 in another weldable bottle or bag, and weld the bottle or bag to the harvest line.

[1217] Table 38. Culture medium used for step 3 from day 4 to day 6 .

[1218]

[1219] e. Once the 3.4L culture medium has been replaced in the vessel, remove 0.1L of culture medium through the 40µm pipette. Track the volume removed by monitoring the liquid level on the vessel screen.

[1220] f. Add 50 mL of the culture medium from step 3 to the reactor.

[1221] g. Prepare another weldable bottle or bag using 40 mL of advanced DMEM / F12 medium + 10 mL of StemFit differentiation medium, and weld the bottle or bag to the harvest pipeline.

[1222] h. Transfer 50 mL of DMEM / F12 medium + StemFit differentiation medium into the reactor.

[1223] i. Clamp the harvesting line and the extraction line.

[1224] j. Incubate and culture the cells at 37°C, 5% CO2, and 23 rpm for two days.

[1225] 3.10 Day 6: Hematopoietic redifferentiation begins—the transitional stage from endothelial cells to hematopoietic cells.

[1226] a. On day 6, preheat the advanced DMEM / F12 medium and StemFit differentiation medium at RT.

[1227] b. On day 6, record the images as in step 3.8b.

[1228] c. Transfer 2720 mL of advanced DMEM / F12 medium + 680 mL of StemFit differentiation medium to a weldable bottle or bag. Alternatively, aliquot the volume into two bottles or bags as in step 3.8ci. Remove the medium from the reactor through a 40 µm pick-up line as in step 3.8de, and add fresh medium to the reactor via a harvest line.

[1229] d. Prepare 50 mL of the culture medium from day 6 to day 8 in another weldable bottle or bag, and weld the bottle or bag to the harvest line.

[1230] Table 39. Culture medium used for step 4 from day 6 to day 8 .

[1231]

[1232] e. Once the 3.4L culture medium has been replaced in the vessel, remove 0.1L of culture medium through the 40µm pipette. Track the volume removed by monitoring the liquid level on the vessel screen.

[1233] f. Add 50 mL of the culture medium from step 4 to the reactor.

[1234] g. Prepare another weldable bottle or bag using 40 mL of advanced DMEM / F12 medium + 10 mL of StemFit differentiation medium, and weld the bottle or bag to the harvest pipeline.

[1235] h. Transfer 50 mL of DMEM / F12 medium + StemFit differentiation medium into the reactor.

[1236] i. Clamp the harvesting line and the extraction line.

[1237] j. Incubate and culture the cells at 37°C, 5% CO2, and 23 rpm for two days.

[1238] 3.11 Day 8: The Continued Beginning of Hematopoietic Redifferentiation—The Transition from Endothelial Cells to Hematopoietic Cells

[1239] a. On day 8, preheat the advanced DMEM / F12 medium and StemFit differentiation medium at RT and record images as in step 3.8b.

[1240] b. In another weldable bottle or bag, prepare approximately 2 L of the culture medium from step 5 for use from day 8 to day 10.

[1241] Table 40. Culture medium used in step 5 for days 8 to 10 .

[1242]

[1243] c. Connect the bottle or bag containing the culture medium from step 5 to the reactor harvest line.

[1244] d. Weld the empty 2L waste bottle to the 4µm draw tube waste line.

[1245] e. During the 2-day process, the culture medium was simultaneously removed from the reactor through a 4µm pick-up line and added back to the reactor via a harvest line at a rate of 0.69 mL / min (1 L / day) until 2 L of culture medium was replaced.

[1246] Day 10, March 12: Hematopoietic stem cells (iCD34 / iHSC) were harvested and cells were prepared for flow cytometry analysis and cryopreservation, as follows:

[1247] a. Record the image on day 10 as in step 3.8b.

[1248] b. Warm 40 mL of advanced DMEM / F12 medium free of cytokines and growth factors.

[1249] c. Process single-cell fractionation as follows:

[1250] i. Use the harvest line pump to remove the entire 2.5L cell volume from the reactor.

[1251] ii. Transfer the entire cell volume of the 500 mL aliquot into a 500 mL centrifuge container and centrifuge at 100 g for 1 minute at brake position 6. Reserve the precipitate for dissociation in step 3.12d, and dissociate the supernatant containing single cells in the following steps.

[1252] iii. Transfer the supernatant containing the single cell to a new 500 mL centrifuge container. Rotate at 300 g for 5 minutes at brake setting 9.

[1253] iv. After centrifugation, collect 500 µL of the cell supernatant for metabolic samples. Remove the remaining supernatant and resuspend the single-cell pellet in 5 mL of fresh, high-quality DMEM / F12 basal medium without added cytokines or growth factors.

[1254] v. Use NC-200 to perform cell counting.

[1255] d. Process cluster cell fractionation as follows:

[1256] i. Add 1 mL of collagenase to each precipitate from step 3.12c.ii. Resuspend and combine in a 50 mL conical tube (total volume of collagenase = 5 mL). Add another 5 mL of collagenase to the solution and mix 5 to 10 times. Incubate at 37°C for 20 minutes.

[1257] ii. After incubation, add an equal volume of high-quality DMEM / F12 medium without added cytokines or growth factors to the tube. Centrifuge at 300g for 5 minutes at brake setting 9.

[1258] iii. Remove the supernatant, add 5 mL of TrypLE, and mix the solution. Incubate at 37°C for 5 minutes.

[1259] iv. Add 8 mL of high-quality DMEM / F12 medium without added cytokines or growth factors, and mix the solution 5 to 10 times or until the clusters are no longer visible.

[1260] v. If clumping is observed after dissociation, pass the cells through a 70µm filter.

[1261] vi. Centrifuge at 300g for 5 minutes at brake position 9.

[1262] vii. Resuspend in 10 mL of high-grade DMEM / F12 medium without added cytokines or growth factors, and perform cell counting using NC-200.

[1263] e. 5.0 × 10 6 Each cell was aliquoted and used for flow cytometry analysis using the iHSC group.

[1264] The f.iHSC flow group is shown in Table 34.

[1265] Exemplary success criteria for the g.iHSC redifferentiation step: purity of each input iPSC >70% lineage-CD34+ cells and fold expansion >5.

[1266] This approach enables the γδ T-iPSC line to redifferentiate into hematopoietic stem cells (HSCs) using a serum-free, feeder-free (SFFF) three-dimensional (3D) culture method in a 3L vertical wheel bioreactor with multiple RPMs, resulting in improvements in cell cluster formation, morphology, cell growth (e.g., metabolism), and purity. The same applies to perfusion within the bioreactor system.

[1267] Figures 3 to 6 This demonstrates a 3D-VW bioreaction protocol for redifferentiating iPSCs into iHSCs, resulting in high purity, high cell viability, high yield, and sufficient fold expansion. The protocol also yields iγδ T cells with purity meeting exemplary success criteria and fold expansion consistent with iHSCs generated using 2D methods. These data demonstrate the efficiency and effectiveness of the 3D-VW bioreaction protocol for generating iHSCs from γδ T-iPSCs, such as… Figures 3 to 6 As shown in the image.

[1268] List of Implementation Plans

[1269] The following is a non-exhaustive list of the embodiments covered by this invention.

[1270] A1. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1271] Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days;

[1272] The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days.

[1273] On day 8 of culture, all cells in the culture were retained while the culture medium was removed from the culture, a process that continued for approximately 2 days; and

[1274] Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

[1275] A2. The method according to item A1, wherein the tissue culture dish has a non-traditional bioreactor geometry.

[1276] A3. The method according to item A1 or item A2, wherein the iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[1277] A4. The method according to any one of items A1 to A3, wherein the tissue culture dish with a vertical wheel further comprises a first culture medium containing StemFit ® The medium consists of one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Crmon 1, Enrica, polyamine, and trans-ISRIB (CEPT).

[1278] The BMP4 and rhVEGF are present in a 1:1 ratio, and

[1279] The first culture medium does not contain antibiotics.

[1280] A5. The method according to any one of items A1 to A4, wherein the method further comprises replacing the used culture medium in the tissue culture dish including the vertical wheel on each of the second, fourth, and sixth days of culture lasting approximately eight days, wherein the step of replacing the used culture medium comprises:

[1281] The used culture medium was removed from the tissue culture dish, including the vertical wheel, using a 40µm cell retention filter, and wash medium was added at a pump flow rate of 30 mL / min. The wash medium contained advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation culture medium;

[1282] In the bioreactor, approximately 3 L to approximately 3.5 L of the wash culture medium is replaced, and 0.1 L of the wash culture medium is removed from the tissue culture dish, which includes a vertical wheel, using a 40 µm cell retention filter; and

[1283] Add approximately 50 mL of the washing medium and approximately 50 mL of the culture medium to the tissue culture dish including the vertical wheel.

[1284] A6. The method according to any one of items A1 to A5, wherein the iPSCs dissociate into single cells on day 7 of culture.

[1285] A7. The method according to any one of items A1 to A6, wherein the step of retaining all cells includes retaining clusters, half-clusters, and single cells.

[1286] A8. The method according to any one of items A1 to A7, wherein the step of retaining all cells comprises removing the used culture medium from the tissue culture dish including the vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 days to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium comprises advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them,

[1287] The fresh culture medium mentioned therein does not contain antibiotics.

[1288] A9. The method according to any one of items A1 to A8, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

[1289] A10. The method according to any one of items A1 to A9, wherein the harvesting step comprises:

[1290] a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet;

[1291] b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics.® In the differentiation culture medium;

[1292] c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension.

[1293] d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the cell suspension containing the combination. ® Differentiation culture medium;

[1294] e) Centrifuge the combined cell suspension to produce a third cell precipitate;

[1295] f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension;

[1296] g) Mix the fourth cell suspension until the cell clusters are no longer visible;

[1297] h) If cell clusters are visible after the mixing step, optionally pass the fourth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[1298] i) Centrifuge the fourth cell suspension.

[1299] A11. The method according to any one of items A1 to A10, wherein the cells are cultured under normoxic conditions.

[1300] A12. The method according to any one of items A1 to A11, wherein the method further includes further purification and / or separation of the redifferentiated iHSC.

[1301] A13. The method according to any one of items A1 to A12, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

[1302] A14. The method according to any one of items A1 to A13, wherein determining the purity of the redifferentiated iHSC comprises determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a by flow cytometry or an equivalent method.

[1303] A15. The method according to item A14, wherein the pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[1304] B1. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1305] Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days;

[1306] The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days.

[1307] Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

[1308] B2. The method according to item B1, wherein the tissue culture dish has a non-traditional bioreactor geometry.

[1309] B3. The method according to item B1 or B2, wherein the iPSC is approximately 1.0 × 10 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[1310] B4. The method according to any one of items B1 to B3, wherein the tissue culture dish with a vertical wheel further comprises a first culture medium containing StemFit. ® The medium consists of one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Crmon 1, Enrica, polyamine, and trans-ISRIB (CEPT).

[1311] The BMP4 and rhVEGF are present in a 1:1 ratio, and

[1312] The first culture medium does not contain antibiotics.

[1313] B5. The method according to any one of items B1 to B4, wherein the method further comprises replacing the used culture medium in the tissue culture dish including the vertical wheel on each of the 2nd, 4th, 6th and 8th days of culture lasting about 8 days, wherein the step of replacing the used culture medium comprises:

[1314] a) Allow the cell aggregates to settle in the tissue culture dish, including the vertical wheel, for approximately 3 to 5 minutes;

[1315] b) Collect 80% of the used culture medium from the tissue culture dish including the vertical wheel;

[1316] c) Centrifuge the collected culture medium to obtain a cell pellet;

[1317] d) Resuspend the cell pellet in StemFit. ® In a differentiation culture medium, a cell suspension is obtained; and

[1318] e) Add the cell suspension and fresh culture medium to the tissue culture dish including the vertical wheel.

[1319] B6. The method according to any one of items B1 to B5, wherein the step of replacing the used culture medium on the 2nd, 4th, and 6th days of cultivation includes:

[1320] i) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a cell pellet; resuspend the cell pellet in StemFit. ® The cells are then added to a differentiation medium to obtain a cell suspension; and the cell suspension and fresh medium are added to the tissue culture dish including a vertical wheel, or...

[1321] ii) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for about 3 to about 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a first cell pellet and a first supernatant; resuspend the first cell pellet in StemFit. ®The first cell pellet, resuspended in differentiation medium, is added to the tissue culture dish including a vertical wheel; the supernatant is centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet is resuspended in StemFit medium. ® The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to the tissue culture dish containing the vertical wheel.

[1322] B7. The method according to any one of items B1 to B6, wherein the fresh culture medium added on the second day of culture comprises:

[1323] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[1324] ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them,

[1325] The VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and

[1326] The second culture medium does not contain antibiotics.

[1327] B8. The method according to any one of items B1 to B7, wherein the iPSCs dissociate into single cells on day 7 of culture.

[1328] B9. The method according to any one of items B1 to B8, wherein the step of harvesting redifferentiated iHSCs includes retaining clusters, hemiclusters, and single cells.

[1329] B10. The method according to any one of items B1 to B9, wherein the harvesting step includes retaining all cells in the tissue culture dish including the vertical wheel.

[1330] B11. The method according to any one of items B1 to B10, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

[1331] B12. The method according to any one of items B1 to B11, wherein the step of harvesting the redifferentiated iHSCs further comprises:

[1332] a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet;

[1333] b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant;

[1334] c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics;

[1335] d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension;

[1336] e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension.

[1337] f) Centrifuge the first cell suspension to produce a third cell precipitate;

[1338] g) The third cell pellet is resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics is added to produce a second cell suspension.

[1339] h) Mix the second cell suspension until the cell clusters are no longer visible;

[1340] i) If cell clusters are visible after the mixing step, the second cell suspension may optionally be passed through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[1341] j) Centrifuge the second cell suspension.

[1342] B13. The method according to any one of items B1 to B12, wherein the cells are cultured under normoxic conditions.

[1343] B14. The method according to any one of items B1 to B13, the method further comprising further purification and / or separation of the redifferentiated iHSCs.

[1344] B15. The method according to any one of items B1 to B14, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

[1345] B16. The method according to any one of items B1 to B15, wherein determining the purity of the redifferentiated iHSC comprises determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a by flow cytometry or an equivalent method.

[1346] B17. The method according to item B16, wherein the pedigree markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[1347] B18. One or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising the steps according to any one of items A1 to B17.

[1348] C1. One or more cells, said one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1349] Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days;

[1350] The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days.

[1351] On day 8 of culture, all cells in the culture were retained while the culture medium was removed from the culture, a process that continued for approximately 2 days; and

[1352] Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

[1353] C2. One or more cells as described in item C1, wherein the tissue culture dish has a non-traditional bioreactor geometry.

[1354] C3. One or more cells as described in item C1 or C2, wherein the iPSCs are approximately 1.0 × 10⁻⁶. 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[1355] C4. One or more cells according to any one of items C1 to C3, including the tissue culture dish with a vertical wheel further comprising a first culture medium containing StemFit. ® The medium consists of one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Crmon 1, Enrica, polyamine, and trans-ISRIB (CEPT).

[1356] The BMP4 and rhVEGF are present in a 1:1 ratio, and

[1357] The first culture medium does not contain antibiotics.

[1358] C5. One or more cells according to any one of items C1 to C4, wherein the method further comprises replacing the used culture medium in the tissue culture dish including the vertical wheel on each of the 2nd, 4th and 6th days of culture lasting about 8 days, wherein the step of replacing the used culture medium includes:

[1359] The used culture medium was removed from the tissue culture dish, including the vertical wheel, using a 40µm cell retention filter, and wash medium was added at a pump flow rate of 30 mL / min. The wash medium contained advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation culture medium;

[1360] In the bioreactor, approximately 3 L to approximately 3.5 L of the wash culture medium is replaced, and 0.1 L of the wash culture medium is removed from the tissue culture dish, which includes a vertical wheel, using a 40 µm cell retention filter; and

[1361] Add approximately 50 mL of the washing medium and approximately 50 mL of the culture medium to the tissue culture dish including the vertical wheel.

[1362] C6. One or more cells according to any one of items C1 to C5, wherein the iPSCs dissociate into single cells on day 7 of culture.

[1363] C7. One or more cells according to any one of items C1 to C6, wherein the step of retaining all cells includes retaining clusters, half-clusters, and single cells.

[1364] C8. One or more cells according to any one of items C1 to C7, wherein the step of retaining all cells comprises removing used culture medium from the tissue culture dish including a vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 days to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium comprises advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them,

[1365] The fresh culture medium mentioned therein does not contain antibiotics.

[1366] C9. One or more cells according to any one of items C1 to C8, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

[1367] C10. One or more cells according to any one of items C1 to C9, wherein the harvesting step comprises:

[1368] a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet;

[1369] b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium;

[1370] c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension.

[1371] d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the cell suspension containing the combination. ® Differentiation culture medium;

[1372] e) Centrifuge the combined cell suspension to produce a third cell precipitate;

[1373] f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension;

[1374] g) Mix the fourth cell suspension until the cell clusters are no longer visible;

[1375] h) If cell clusters are visible after the mixing step, optionally pass the fourth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[1376] i) Centrifuge the fourth cell suspension.

[1377] C11. One or more cells according to any one of items C1 to C10, wherein the cells are cultured under normoxic conditions.

[1378] C12. One or more cells according to any one of items C1 to C11, further comprising further purification and / or isolation of the redifferentiated iHSCs.

[1379] C13. One or more cells according to any one of items C1 to C12, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

[1380] C14. One or more cells according to any one of items C1 to C13, wherein determining the purity of the redifferentiated iHSC comprises determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a by flow cytometry or an equivalent method.

[1381] C15. One or more cells as described in item C14, wherein the lineage markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[1382] D1. One or more cells, said one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1383] Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days;

[1384] The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days.

[1385] Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

[1386] D2. One or more cells as described in item D1, wherein the tissue culture dish has a non-traditional bioreactor geometry.

[1387] D3. One or more cells as described in item D1 or D2, wherein the iPSCs are approximately 1.0 × 10⁻⁶. 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

[1388] D4. One or more cells according to any one of items D1 to D3, including the tissue culture dish with a vertical wheel further comprising a first culture medium containing StemFit. ® The medium consists of one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Crmon 1, Enrica, polyamine, and trans-ISRIB (CEPT).

[1389] The BMP4 and rhVEGF are present in a 1:1 ratio, and

[1390] The first culture medium does not contain antibiotics.

[1391] D5. One or more cells according to any one of items D1 to D4, wherein the method further comprises replacing the used culture medium in the tissue culture dish including the vertical wheel on each of the 2nd, 4th, 6th and 8th days of culture lasting approximately 8 days, wherein the step of replacing the used culture medium comprises:

[1392] a) Allow the cell aggregates to settle in the tissue culture dish, including the vertical wheel, for approximately 3 to 5 minutes;

[1393] b) Collect 80% of the used culture medium from the tissue culture dish including the vertical wheel;

[1394] c) Centrifuge the collected culture medium to obtain a cell pellet;

[1395] d) Resuspend the cell pellet in StemFit. ® In a differentiation culture medium, a cell suspension is obtained; and

[1396] e) Add the cell suspension and fresh culture medium to the tissue culture dish including the vertical wheel.

[1397] D6. One or more cells according to any one of items D1 to D5, wherein the step of replacing the used culture medium on days 2, 4, and 6 of culture includes:

[1398] i) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a cell pellet; resuspend the cell pellet in StemFit. ® The cells are then added to a differentiation medium to obtain a cell suspension; and the cell suspension and fresh medium are added to the tissue culture dish including a vertical wheel, or...

[1399] ii) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for about 3 to about 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a first cell pellet and a first supernatant; resuspend the first cell pellet in StemFit. ® The first cell pellet, resuspended in differentiation medium, is added to the tissue culture dish including a vertical wheel; the supernatant is centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet is resuspended in StemFit medium. ® The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to the tissue culture dish containing the vertical wheel.

[1400] D7. One or more cells according to any one of items D1 to D6, wherein the fresh culture medium added on day 2 of culture comprises:

[1401] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[1402] ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them,

[1403] The VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and

[1404] The second culture medium does not contain antibiotics.

[1405] D8. One or more cells according to any one of items D1 to D7, wherein the iPSCs dissociate into single cells on day 7 of culture.

[1406] D9. One or more cells according to any one of items D1 to D8, wherein the step of harvesting redifferentiated iHSCs includes retaining clusters, hemiclusters, and single cells.

[1407] D10. One or more cells according to any one of D1 to D9, wherein the step of retaining all cells comprises removing used culture medium from the tissue culture dish including a vertical wheel using a 4µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 days to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium comprises advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them,

[1408] The fresh culture medium mentioned therein does not contain antibiotics.

[1409] D11. One or more cells according to any one of items D1 to D10, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

[1410] D12. One or more cells according to any one of items D1 to D11, wherein the step of harvesting redifferentiated iHSCs includes:

[1411] a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet;

[1412] b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant;

[1413] c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics;

[1414] d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension;

[1415] e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension.

[1416] f) Centrifuge the first cell suspension to produce a third cell precipitate;

[1417] g) The third cell pellet is resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics is added to produce a second cell suspension.

[1418] h) Mix the second cell suspension until the cell clusters are no longer visible;

[1419] i) If cell clusters are visible after the mixing step, the second cell suspension may optionally be passed through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and

[1420] j) Centrifuge the second cell suspension.

[1421] D13. One or more cells according to any one of items D1 to D12, wherein the cells are cultured under normoxic conditions.

[1422] D14. One or more cells according to any one of items D1 to D13, further comprising further purification and / or isolation of the redifferentiated iHSCs.

[1423] D15. One or more cells according to any one of items D1 to D14, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

[1424] D16. One or more cells according to any one of items D1 to D15, wherein the one or more cells have high purity, viability, fold expansion and the potential to redifferentiate into immune effector cells, said immune effector cells including iPSC-derived γδ (iγδ) T cells.

[1425] D17. One or more cells according to any one of items D1 to D16, wherein the step of determining the purity of the iHSC comprises: determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a, optionally all of them, via flow cytometry or an equivalent method.

[1426] D18. One or more cells according to any one of items D1 to D17, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

[1427] E1. A composition comprising one or more cells according to any one of items C1 to D18.

[1428] F1. Use of one or more cells according to any one of items C1 to D18 in the preparation of cells for treating lesions or diseases, in the preparation of lymphocytes, in a bioreactor, or in in vitro drug screening for tissue engineering or diseases.

[1429] G1. A system for performing a method of de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising the steps of any one of claims A1 to B17, wherein the vertical wheel is perpendicular or not perpendicular to the vessel.

[1430] H1. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1431] Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days;

[1432] Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days;

[1433] A device for retaining all cells in a culture on day 8 of culture while removing the culture medium from the culture, for approximately 2 days; and

[1434] A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

[1435] 1. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1436] Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days;

[1437] Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days;

[1438] A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

[1439] J1. An iHSC, said iHSC being generated according to the method of item H1 or item I1.

[1440] K1. One or more cells, said one or more cells being obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1441] Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days;

[1442] Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days;

[1443] A device for retaining all cells in a culture on day 8 of culture while removing the culture medium from the culture, for approximately 2 days; and

[1444] A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

[1445] L1. One or more cells, said one or more cells being obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδT cell-derived induced pluripotent stem cells (iPSCs), said method comprising:

[1446] Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days;

[1447] Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days;

[1448] A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

[1449] M1. A method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[1450] i) Obtaining one or more human induced pluripotent stem cells (iPSCs), and said method further comprising one or more of the following steps:

[1451] a) In a tissue culture dish including a vertical wheel, incubate the iPSCs in a first culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, with stirring at about 10 rpm to 60 rpm, preferably 40 rpm. Then, remove the entire cell volume from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume.

[1452] The first culture medium contains StemFit ® The first medium contains one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and Rho kinase inhibitor (ROCK inhibitor), and a mixture of Crman 1, Enrica, polyamine, and trans-ISRIB (CEPT), wherein the first medium does not contain antibiotics.

[1453] b) Centrifuge the first cell suspension to produce a first cell precipitate;

[1454] c) Resuspending the first cell pellet in a second culture medium to form a second cell suspension, wherein the second culture medium comprises:

[1455] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or

[1456] ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™-34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them,

[1457] The VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and the second culture medium does not contain antibiotics.

[1458] d) The second cell suspension is cultured in the tissue culture dish including the vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[1459] e) Stop stirring for about 5 minutes, during which the cell aggregates settle in the second cell suspension;

[1460] f) Collect approximately 80% of the top volume of the second cell suspension from the tissue culture dish including the vertical wheel;

[1461] g) Centrifuge the collected second cell suspension to form a second cell pellet and resuspend the second cell pellet in approximately 1 mL of StemFit solution. ® Differentiation solution and / or StemPro ™ -34 SFM in one of them;

[1462] h) The resuspended second cell pellet is added to a third culture medium to form a third cell suspension, wherein the third culture medium comprises:

[1463] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[1464] ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[1465] Furthermore, the third culture medium does not contain antibiotics;

[1466] i) The third cell suspension is placed into the tissue culture dish including a vertical wheel, which contains the remaining second cell suspension, thereby forming a fourth cell suspension;

[1467] j) The fourth cell suspension is cultured in the tissue culture dish including the vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[1468] k) Stop stirring for about 5 minutes; during this time, the cell aggregates settle in the cell suspension.

[1469] l) Collect approximately 80% of the fourth cell suspension and centrifuge the collected fourth cell suspension to form a third cell pellet, and resuspend the third cell pellet in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM in one of them;

[1470] m) Dissolve in approximately 1 mL of StemFit ® Differentiation solution and StemPro ™ The third cell pellet in one of the -34 SFMs is suspended in a fourth culture medium to form a fifth cell suspension, wherein the fourth culture medium comprises:

[1471] i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or

[1472] ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™-34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them,

[1473] Furthermore, the fourth culture medium does not contain antibiotics;

[1474] n) The fifth cell suspension is placed into the tissue culture dish including a vertical wheel, which contains the remaining fourth cell suspension, thereby forming a sixth cell suspension;

[1475] o) The sixth cell suspension is cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[1476] p) Repeat steps k) to o); optionally, wherein the sixth cell suspension is centrifuged and the supernatant from the centrifuged sixth cell suspension is centrifuged, and the resulting cell pellet is resuspended in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM, and combined with a seventh cell suspension obtained by resuspending the cell pellet from the first centrifugation to form an eighth cell suspension, the eighth cell suspension is then cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days.

[1477] q) Remove all of the eighth cell suspension from the tissue culture dish including the vertical wheel, and harvest EB and iHSC from the eighth cell suspension by centrifugation to produce a fourth cell pellet and a first supernatant;

[1478] r) Collect the first supernatant;

[1479] s) The first supernatant is centrifuged to produce a fifth cell pellet, and the fifth cell pellet is resuspended in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium comprises advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells of the ninth cell suspension comprise iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a;

[1480] t) The fourth cell pellet from step q) is resuspended in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension;

[1481] u) Add the fifth culture medium to the tenth cell suspension;

[1482] v) Centrifuge the tenth cell suspension to produce a sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce an eleventh cell suspension.

[1483] w) Add the additional fifth culture medium to the eleventh cell suspension and mix;

[1484] x) The eleventh cell suspension is centrifuged to form a seventh cell pellet, and the seventh cell pellet is resuspended in another fifth culture medium to form a twelfth cell suspension.

[1485] y) If cell clumps are present in the twelfth cell suspension, the twelfth cell suspension may optionally be passed through a cell filter to obtain iHSC, wherein the cell filter has a 70 µm mesh size;

[1486] z) Centrifuge the twelfth cell suspension;

[1487] aa) No serum is introduced in any of the steps a)-z);

[1488] bb) No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-aa).

[1489] cc) Determine the purity of the iHSC from the first supernatant of step s) and the iHSC from step y), the purity being defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and

[1490] dd) to obtain the iHSC generated from scratch.

[1491] N1. A method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, the method comprising one or more of the following steps:

[1492] i) Obtaining one or more human induced pluripotent stem cells (iPSCs), and said method further comprising one or more of the following steps:

[1493] a) Inoculating the iPSCs, wherein the inoculation is performed by suspending the iPSCs in a first culture medium in a tissue culture dish including a vertical wheel to generate a first cell suspension, wherein the first culture medium contains StemFit. ® Basic 04 medium and StemFit ® One of the following: Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromogen 1, enricane, polyamine and trans-ISRIB (CEPT).

[1494] Furthermore, the first culture medium does not contain antibiotics;

[1495] b) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days, during which embryoid bodies (EB) are generated.

[1496] c) Remove the first culture medium from the tissue culture dish including the vertical wheel and add wash medium at a rate of about 30 mL / min, the wash medium containing advanced DMEM / F12 medium and StemFit in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation culture medium;

[1497] d) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of the wash medium in the tissue culture dish including the vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of the wash medium from the tissue culture dish including the vertical wheel;

[1498] e) Add approximately 50 mL of wash medium and approximately 50 mL of a second medium to the tissue culture dish including the vertical wheel, wherein the second medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ®The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax™ supplements, wherein the second medium does not contain antibiotics;

[1499] f) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[1500] g) Remove the second culture medium from the tissue culture dish including the vertical wheel and add the wash medium at a rate of about 30 mL / min;

[1501] h) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of the wash medium in the tissue culture dish including the vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of the wash medium from the tissue culture dish including the vertical wheel;

[1502] i) Add approximately 50 mL of wash medium and approximately 50 mL of third medium to a tissue culture dish including a vertical wheel, wherein the third medium contains advanced DMEM / F12 medium and StemFit medium present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the third culture medium does not contain antibiotics;

[1503] j) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[1504] k) Remove the third culture medium from the tissue culture dish including the vertical wheel and add the wash medium at a rate of about 30 mL / min;

[1505] l) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of the wash medium in the tissue culture dish including the vertical wheel at a pump flow rate of 30mL / min and remove 0.1L of the wash medium from the tissue culture dish including the vertical wheel;

[1506] m) Add approximately 50 mL of wash medium and approximately 50 mL of a fourth medium to the tissue culture dish including the vertical wheel, wherein the fourth medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fourth culture medium does not contain antibiotics;

[1507] n) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days;

[1508] o) Using a 4µm cell retention filter, at a pump flow rate of approximately 0.69 mL / min (1 L / day), simultaneously remove the fourth culture medium from the tissue culture dish including the vertical wheel and add the fifth culture medium to the tissue culture dish including the vertical wheel within approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days, until 2 L of culture medium has been replaced. The fifth culture medium contains advanced DMEM / F12 medium and StemFit at a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fifth culture medium does not contain antibiotics;

[1509] p) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet;

[1510] q) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth fac...

Claims

1. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days; The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days. On day 8 of culture, all cells in the culture were retained while the culture medium was removed from the culture, a process that continued for approximately 2 days; and Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

2. The method of claim 1, wherein the tissue culture dish has a non-traditional bioreactor geometry.

3. The method of claim 1 or claim 2, wherein the iPSCs are seeded at a density of about 1.0 x 10 5 cells / mL to about 1.8 x 10 5 cells / mL.

4. The method of any one of claims 1-3, wherein the tissue culture dish comprising vertical wheels further comprises a first culture medium comprising StemFit ® Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of a Rho kinase inhibitor (ROCK inhibitor) and a mix of Kavalenka, Lenabre, polyamine, and trans-ISRIB (CEPT). The BMP4 and rhVEGF are present in a 1:1 ratio, and The first culture medium does not contain antibiotics.

5. The method according to any one of claims 1 to 4, wherein the method further comprises replacing the used culture medium in the tissue culture dish including the vertical wheel on each of the second, fourth, and sixth days of culture lasting approximately eight days, wherein the step of replacing the used culture medium comprises: The used culture medium was removed from the tissue culture dish, including the vertical wheel, using a 40µm cell retention filter, and wash medium was added at a pump flow rate of 30 mL / min. The wash medium contained advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation culture medium; In the bioreactor, approximately 3 L to approximately 3.5 L of the wash culture medium is replaced, and 0.1 L of the wash culture medium is removed from the tissue culture dish, which includes a vertical wheel, using a 40 µm cell retention filter; and Add approximately 50 mL of the washing medium and approximately 50 mL of the culture medium to the tissue culture dish including the vertical wheel.

6. The method according to any one of claims 1 to 5, wherein the iPSCs dissociate into single cells on day 7 of culture.

7. The method according to any one of claims 1 to 6, wherein the step of retaining all cells includes retaining clusters, half-clusters, and single cells.

8. The method according to any one of claims 1 to 7, wherein the step of retaining all cells comprises removing used culture medium from the tissue culture dish including the vertical wheel using a 4 µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 days to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium comprises advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, The fresh culture medium mentioned therein does not contain antibiotics.

9. The method according to any one of claims 1 to 8, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

10. The method according to any one of claims 1 to 9, wherein the harvesting step comprises: a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet; b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium; c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension. d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the cell suspension containing the combination. ® Differentiation culture medium; e) Centrifuge the combined cell suspension to produce a third cell precipitate; f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension; g) Mix the fourth cell suspension until the cell clusters are no longer visible; h) If cell clusters are visible after the mixing step, optionally pass the fourth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and i) Centrifuge the fourth cell suspension.

11. The method according to any one of claims 1 to 10, wherein the cells are cultured under normoxic conditions.

12. The method according to any one of claims 1 to 11, the method further comprising further purification and / or separation of the redifferentiated iHSCs.

13. The method according to any one of claims 1 to 12, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

14. The method according to any one of claims 1 to 13, wherein determining the purity of the redifferentiated iHSC comprises determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a by flow cytometry or an equivalent method.

15. The method of claim 14, wherein the phylogenetic markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

16. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days; The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days. Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

17. The method of claim 16, wherein the tissue culture dish has a non-traditional bioreactor geometry.

18. The method of claim 16 or claim 17, wherein the iPSC is at approximately 1.0 × 10⁻⁶. 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

19. The method according to any one of claims 16 to 18, wherein the tissue culture dish with the vertical wheel further comprises a first culture medium containing StemFit ® The medium consists of one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Crmon 1, Enrica, polyamine, and trans-ISRIB (CEPT). The BMP4 and rhVEGF are present in a 1:1 ratio, and The first culture medium does not contain antibiotics.

20. The method according to any one of claims 16 to 19, wherein the method further comprises replacing the used culture medium in the tissue culture dish including the vertical wheel on each of the 2nd, 4th, 6th, and 8th days of culture lasting approximately 8 days, wherein the step of replacing the used culture medium comprises: a) Allow the cell aggregates to settle in the tissue culture dish, including the vertical wheel, for approximately 3 to 5 minutes; b) Collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; c) Centrifuge the collected culture medium to obtain a cell pellet; d) Resuspend the cell pellet in StemFit. ® In the differentiation culture medium, a cell suspension is obtained; as well as e) Add the cell suspension and fresh culture medium to the tissue culture dish including the vertical wheel.

21. The method according to any one of claims 15 to 20, wherein the step of replacing the used culture medium on the second, fourth, and sixth days of cultivation comprises: i) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a cell pellet; resuspend the cell pellet in StemFit. ® In the differentiation culture medium, a cell suspension is obtained; And adding the cell suspension and fresh culture medium to the tissue culture dish including the vertical wheel, or ii) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for about 3 to about 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a first cell pellet and a first supernatant; The first cell pellet was resuspended in StemFit. ® The first cell pellet, resuspended in differentiation medium, is added to the tissue culture dish including a vertical wheel; the supernatant is centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet is resuspended in StemFit medium. ® The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to the tissue culture dish containing the vertical wheel.

22. The method according to any one of claims 15 to 22, wherein the fresh culture medium added on the second day of culture comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them, The VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and The second culture medium does not contain antibiotics.

23. The method according to any one of claims 16 to 22, wherein the iPSCs dissociate into single cells on day 7 of culture.

24. The method according to any one of claims 16 to 23, wherein the step of harvesting redifferentiated iHSCs includes retaining clusters, half-clusters, and single cells.

25. The method according to any one of claims 16 to 24, wherein the harvesting step comprises retaining all cells in the tissue culture dish including the vertical wheel.

26. The method according to any one of claims 16 to 25, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

27. The method according to any one of claims 16 to 26, wherein the step of harvesting the redifferentiated iHSCs further comprises: a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet; b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant; c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics; d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension; e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension. f) Centrifuge the first cell suspension to produce a third cell precipitate; g) The third cell pellet is resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics is added to produce a second cell suspension. h) Mix the second cell suspension until the cell clusters are no longer visible; i) If cell clusters are visible after the mixing step, the second cell suspension may optionally be passed through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and j) Centrifuge the second cell suspension.

28. The method according to any one of claims 16 to 27, wherein the cells are cultured under normoxic conditions.

29. The method according to any one of claims 16 to 28, further comprising purifying and / or isolating the redifferentiated iHSCs.

30. The method according to any one of claims 16 to 29, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

31. The method according to any one of claims 16 to 30, wherein determining the purity of the redifferentiated iHSC comprises determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a by flow cytometry or an equivalent method.

32. The method of claim 31, wherein the phylogenetic markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

33. One or more cells, said one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising the steps of any one of claims 1 to 32.

34. One or more cells, said one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days; The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days. On day 8 of culture, all cells in the culture were retained while the culture medium was removed from the culture, a process that continued for approximately 2 days; and Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

35. One or more cells according to claim 34, wherein the tissue culture dish has a non-traditional bioreactor geometry.

36. One or more cells according to claim 34 or claim 35, wherein the iPSCs are at about 1.0 × 10⁻⁶. 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

37. One or more cells according to any one of claims 34 to 36, wherein the tissue culture dish comprising a vertical wheel further comprises a first culture medium containing StemFit. ® The medium consists of one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Crmon 1, Enrica, polyamine, and trans-ISRIB (CEPT). The BMP4 and rhVEGF are present in a 1:1 ratio, and The first culture medium does not contain antibiotics.

38. One or more cells according to any one of claims 34 to 37, wherein the method further comprises replacing the used culture medium in the tissue culture dish comprising a vertical wheel on each of the second, fourth, and sixth days of culture lasting approximately eight days, wherein the step of replacing the used culture medium comprises: The used culture medium was removed from the tissue culture dish, including the vertical wheel, using a 40µm cell retention filter, and wash medium was added at a pump flow rate of 30 mL / min. The wash medium contained advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation culture medium; In the bioreactor, approximately 3 L to approximately 3.5 L of the wash culture medium is replaced, and 0.1 L of the wash culture medium is removed from the tissue culture dish, which includes a vertical wheel, using a 40 µm cell retention filter; and Add approximately 50 mL of the washing medium and approximately 50 mL of the culture medium to the tissue culture dish including the vertical wheel.

39. One or more cells according to any one of claims 34 to 38, wherein the iPSCs dissociate into single cells on day 7 of culture.

40. One or more cells according to any one of claims 34 to 39, wherein the step of retaining all cells includes retaining clusters, half-clusters, and single cells.

41. One or more cells according to any one of claims 34 to 40, wherein the step of retaining all cells comprises removing used culture medium from the tissue culture dish including a vertical wheel using a 4 µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 days to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium comprises advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, The fresh culture medium mentioned therein does not contain antibiotics.

42. One or more cells according to any one of claims 34 to 41, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 23 rpm.

43. One or more cells according to any one of claims 34 to 42, wherein the harvesting step comprises: a) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet; b) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium; c) Add a first volume of 20 mg / mL collagenase II to the first cell pellet and the second cell pellet, combine the resuspended first cell pellet and the resuspended second cell pellet and add a second volume of 20 mg / mL collagenase II to produce a combined cell suspension. d) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the cell suspension containing the combination. ® Differentiation culture medium; e) Centrifuge the combined cell suspension to produce a third cell precipitate; f) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a fourth cell suspension; g) Mix the fourth cell suspension until the cell clusters are no longer visible; h) If cell clusters are visible after the mixing step, optionally pass the fourth cell suspension through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and i) Centrifuge the fourth cell suspension.

44. One or more cells according to any one of claims 34 to 43, wherein the cells are cultured under normoxic conditions.

45. One or more cells according to any one of claims 34 to 44, further comprising further purification and / or isolation of the redifferentiated iHSCs.

46. ​​One or more cells according to any one of claims 34 to 45, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

47. One or more cells according to any one of claims 34 to 46, wherein determining the purity of the redifferentiated iHSC comprises determining one or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a by flow cytometry or an equivalent method.

48. One or more cells according to claim 47, wherein the lineage markers include all of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

49. One or more cells, said one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Human γδ T cell-derived iPSCs were seeded in tissue culture dishes including vertical wheels, wherein the iPSCs were maintained for up to about 7 days; The iPSCs were redifferentiated into iHSCs by culturing them in the tissue culture dish, which included a vertical wheel, for approximately 8 days. Around day 10 of culture, the redifferentiated iHSCs were harvested into two different cell fractions: single-cell fractions and multi-cell fractions.

50. One or more cells according to claim 49, wherein the tissue culture dish has a non-traditional bioreactor geometry.

51. One or more cells according to claim 49 or claim 50, wherein the iPSCs are at about 1.0 × 10⁻⁶. 5 Cells / mL to approximately 1.8 × 10⁻⁶ to 1.8 × 10⁻⁶. 5 Seed at a density of cells / mL.

52. One or more cells according to any one of claims 49 to 51, wherein the tissue culture dish comprising a vertical wheel further comprises a first culture medium containing StemFit. ® The medium consists of one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and a mixture of Rho kinase inhibitor (ROCK inhibitor) and Crmon 1, Enrica, polyamine, and trans-ISRIB (CEPT). The BMP4 and rhVEGF are present in a 1:1 ratio, and The first culture medium does not contain antibiotics.

53. One or more cells according to any one of claims 49 to 52, wherein the method further comprises replacing the used culture medium in the tissue culture dish including the vertical wheel on each of the 2nd, 4th, 6th, and 8th days of culture lasting about 8 days, wherein the step of replacing the used culture medium comprises: a) Allow the cell aggregates to settle in the tissue culture dish, including the vertical wheel, for approximately 3 to 5 minutes; b) Collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; c) Centrifuge the collected culture medium to obtain a cell pellet; d) Resuspend the cell pellet in StemFit. ® In the differentiation culture medium, a cell suspension is obtained; as well as e) Add the cell suspension and fresh culture medium to the tissue culture dish including the vertical wheel.

54. One or more cells according to any one of claims 49 to 52, wherein the step of replacing the used culture medium on days 2, 4, and 6 of culture comprises: i) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for approximately 3 to 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a cell pellet; resuspend the cell pellet in StemFit. ® In the differentiation culture medium, a cell suspension is obtained; And adding the cell suspension and fresh culture medium to the tissue culture dish including the vertical wheel, or ii) Allow the cell aggregates to settle in the tissue culture dish including the vertical wheel for about 3 to about 5 minutes; collect 80% of the used culture medium from the tissue culture dish including the vertical wheel; centrifuge the collected culture medium to obtain a first cell pellet and a first supernatant; The first cell pellet was resuspended in StemFit. ® The first cell pellet, resuspended in differentiation medium, is added to the tissue culture dish including a vertical wheel; the supernatant is centrifuged to obtain a second cell pellet and a second supernatant; and the second cell pellet is resuspended in StemFit medium. ® The second cell pellet, resuspended in the differentiation medium, and fresh medium were added to the tissue culture dish containing the vertical wheel.

55. One or more cells according to any one of claims 49 to 54, wherein the fresh culture medium added on the second day of culture comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them, The VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and The second culture medium does not contain antibiotics.

56. One or more cells according to any one of claims 49 to 55, wherein the iPSCs dissociate into single cells on day 7 of culture.

57. One or more cells according to any one of claims 49 to 56, wherein the step of harvesting redifferentiated iHSCs includes retaining clusters, half-clusters, and single cells.

58. One or more cells according to any one of claims 49 to 57, wherein the step of retaining all cells comprises removing used culture medium from the tissue culture dish including a vertical wheel using a 4 µm cell retention filter, and adding fresh culture medium to the bioreactor at a rate of about 0.69 mL / min (1 L / day) for about 1.8 days to about 2.2 days, preferably about 2 days, until 2 L of culture medium has been replaced, wherein the fifth culture medium comprises advanced DMEM / F12 medium and StemFit present in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, The fresh culture medium mentioned therein does not contain antibiotics.

59. One or more cells according to any one of claims 49 to 58, wherein the vertical wheel rotates at a rate of about 10 rpm to about 60 rpm, preferably about 40 rpm.

60. One or more cells according to any one of claims 49 to 59, wherein the step of harvesting redifferentiated iHSCs comprises: a) Remove the entire cell volume containing embryoid bodies (EB) and iHSCs from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet; b) Centrifuge the first supernatant to produce a second supernatant and a second cell precipitate, and remove the second supernatant; c) Resuspend the first and second cell pellets in advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics; d) Add a certain volume of 20 mg / mL collagenase II to the first cell pellet to produce a first cell suspension; e) Add an equal volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics to the first cell suspension. f) Centrifuge the first cell suspension to produce a third cell precipitate; g) The third cell pellet is resuspended in a certain volume of TrypLE and a certain volume of advanced DMEM / F12 medium free of cytokines, growth factors or antibiotics is added to produce a second cell suspension. h) Mix the second cell suspension until the cell clusters are no longer visible; i) If cell clusters are visible after the mixing step, the second cell suspension may optionally be passed through a cell filter to obtain iHSCs, wherein the cell filter has a 70 µm mesh size; and j) Centrifuge the second cell suspension.

61. One or more cells according to any one of claims 49 to 60, wherein the cells are cultured under normoxic conditions.

62. One or more cells according to any one of claims 49 to 61, further comprising further purification and / or isolation of the redifferentiated iHSCs.

63. One or more cells according to any one of claims 49 to 62, wherein the redifferentiated iHSC is an isolated differentiated cell or can be further purified and / or isolated.

64. One or more cells according to any one of claims 49 to 63, wherein the one or more cells have high purity, viability, fold expansion and the potential to redifferentiate into immune effector cells, said immune effector cells including iPSC-derived γδ (iγδ) T cells.

65. One or more cells according to any one of claims 49 to 64, wherein the step of determining the purity of the iHSC comprises: One or more of the lineage markers CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a, or all of them, may be determined by flow cytometry or equivalent methods.

66. One or more cells according to any one of claims 49 to 65, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a.

67. A composition comprising one or more cells according to any one of claims 33 to 66.

68. Use of one or more cells according to any one of claims 38 to 66 in the preparation of cells for treating lesions or diseases, in the preparation of lymphocytes, in a bioreactor, or in in vitro drug screening for tissue engineering or diseases.

69. A system for performing a method of de novo generation of hematopoietic stem cells (iHSCs) under serum-free and feeder-free culture conditions, the hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), the method comprising the steps of any one of claims 1 to 32, wherein the vertical wheel is perpendicular or not perpendicular to the vessel.

70. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days; Device for retaining all cells in a culture on day 8 of culture while removing the culture medium from the culture for approximately 2 days; as well as A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

71. A method for de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free culture conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days; A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

72. An iHSC, said iHSC being generated by the method of claim 38 or claim 39.

73. One or more cells, said one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days; Device for retaining all cells in a culture on day 8 of culture while removing the culture medium from the culture for approximately 2 days; as well as A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

74. One or more cells, said one or more cells obtained from a method of de novo generation of hematopoietic stem cells (iHSCs) in a tissue culture dish under serum-free and feeder-free conditions, said hematopoietic stem cells (iHSCs) being derived from human γδ T cell-derived induced pluripotent stem cells (iPSCs), said method comprising: Apparatus for seeding human γδ T cell-derived iPSCs in a tissue culture dish including a vertical wheel, wherein the iPSCs are maintained for up to about 7 days; Apparatus for redifferentiating iPSCs into iHSCs by culturing them in a tissue culture dish including a vertical wheel for approximately 8 days; A device for harvesting redifferentiated iHSCs into two different cell fractions, namely single-cell fractions and multi-cell fractions, at approximately day 10 of culture.

75. A method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) Obtaining one or more human induced pluripotent stem cells (iPSCs), and said method further comprising one or more of the following steps: a) In a tissue culture dish including a vertical wheel, incubate the iPSCs in a first culture medium for about 1.8 days to about 2.2 days, preferably about 2 days, with stirring at about 10 rpm to 60 rpm, preferably 40 rpm. Then, remove the entire cell volume from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume. The first culture medium contains StemFit ® The first medium contains one of the following: Basic 04 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and Rho kinase inhibitor (ROCK inhibitor), and a mixture of Crman 1, Enrica, polyamine, and trans-ISRIB (CEPT), wherein the first medium does not contain antibiotics. b) Centrifuge the first cell suspension to produce a first cell precipitate; c) Resuspending the first cell pellet in a second culture medium to form a second cell suspension, wherein the second culture medium comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax. ™ One of the supplements, or ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplement, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), as well as L-glutamine and GlutaMax ™ One of them, The VEGF, bFGF, and SCF are present in a ratio of 1:0.625:0.625, and the second culture medium does not contain antibiotics. d) The second cell suspension is cultured in the tissue culture dish including the vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days; e) Stop stirring for about 5 minutes, during which the cell aggregates settle in the second cell suspension; f) Collect approximately 80% of the top volume of the second cell suspension from the tissue culture dish including the vertical wheel; g) Centrifuge the collected second cell suspension to form a second cell pellet and resuspend the second cell pellet in approximately 1 mL of either StemFit® differentiation solution or StemPro™-34 SFM; h) The resuspended second cell pellet is added to a third culture medium to form a third cell suspension, wherein the third culture medium comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them, Furthermore, the third culture medium does not contain antibiotics; i) The third cell suspension is placed into the tissue culture dish including a vertical wheel, which contains the remaining second cell suspension, thereby forming a fourth cell suspension; j) The fourth cell suspension is cultured in the tissue culture dish including the vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days; k) Stop stirring for about 5 minutes; during this time, the cell aggregates settle in the cell suspension. l) Collect approximately 80% of the fourth cell suspension and centrifuge the collected fourth cell suspension to form a third cell pellet, and resuspend the third cell pellet in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM in one of them; m) Dissolve in approximately 1 mL of StemFit ® Differentiation solution and StemPro ™ The third cell pellet in one of the -34 SFMs is suspended in a fourth culture medium to form a fifth cell suspension, wherein the fourth culture medium comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them, Furthermore, the fourth culture medium does not contain antibiotics; n) The fifth cell suspension is placed into the tissue culture dish including a vertical wheel, which contains the remaining fourth cell suspension, thereby forming a sixth cell suspension; o) The sixth cell suspension is cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days; p) Repeat steps k) to o); optionally, wherein the sixth cell suspension is centrifuged and the supernatant from the centrifuged sixth cell suspension is centrifuged, and the resulting cell pellet is resuspended in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM, and combined with a seventh cell suspension obtained by resuspending the cell pellet from the first centrifugation to form an eighth cell suspension, the eighth cell suspension is then cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 40 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days. q) Remove all of the eighth cell suspension from the tissue culture dish including the vertical wheel, and harvest EB and iHSC from the eighth cell suspension by centrifugation to produce a fourth cell pellet and a first supernatant; r) Collect the first supernatant; s) The first supernatant is centrifuged to produce a fifth cell pellet, and the fifth cell pellet is resuspended in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium comprises advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells of the ninth cell suspension comprise iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a; t) The fourth cell pellet from step q) is resuspended in a certain volume of approximately 20 mg / mL type II collagenase to produce the tenth cell suspension; u) Add the fifth culture medium to the tenth cell suspension; v) Centrifuge the tenth cell suspension to produce a sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce an eleventh cell suspension. w) Add the additional fifth culture medium to the eleventh cell suspension and mix; x) The eleventh cell suspension is centrifuged to form a seventh cell pellet, and the seventh cell pellet is resuspended in another fifth culture medium to form a twelfth cell suspension. y) If cell clumps are present in the twelfth cell suspension, the twelfth cell suspension may optionally be passed through a cell filter to obtain iHSC, wherein the cell filter has a 70 µm mesh size; z) Centrifuge the twelfth cell suspension; aa) No serum is introduced in any of the steps a)-z); bb) No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-aa). cc) Determine the purity of the iHSC from the first supernatant of step s) and the iHSC from step y), the purity being defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and dd) to obtain the iHSC generated from scratch.

76. A method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) Obtaining one or more human induced pluripotent stem cells (iPSCs), and said method further comprising one or more of the following steps: a) Inoculating the iPSCs, wherein the inoculation is performed by suspending the iPSCs in a first culture medium in a tissue culture dish including a vertical wheel to generate a first cell suspension, wherein the first culture medium contains StemFit. ® Basic 04 medium and StemFit ® One of the following: Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromogen 1, enricane, polyamine and trans-ISRIB (CEPT). Furthermore, the first culture medium does not contain antibiotics; b) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days, during which embryoid bodies (EB) are generated. c) Remove the first culture medium from the tissue culture dish including the vertical wheel and add wash medium at a rate of about 30 mL / min, the wash medium containing advanced DMEM / F12 medium and StemFit in a ratio of about 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation culture medium; d) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of the wash medium in the tissue culture dish including the vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of the wash medium from the tissue culture dish including the vertical wheel; e) Add approximately 50 mL of wash medium and approximately 50 mL of a second medium to the tissue culture dish including the vertical wheel, wherein the second medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), recombinant human vascular endothelial growth factor (rhVEGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), and one of L-glutamine and GlutaMax™ supplements, wherein the second medium does not contain antibiotics; f) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days; g) Remove the second culture medium from the tissue culture dish including the vertical wheel and add the wash medium at a rate of about 30 mL / min; h) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of the wash medium in the tissue culture dish including the vertical wheel at a pump flow rate of 30mL / min, and remove 0.1L of the wash medium from the tissue culture dish including the vertical wheel; i) Add approximately 50 mL of wash medium and approximately 50 mL of third medium to a tissue culture dish including a vertical wheel, wherein the third medium contains advanced DMEM / F12 medium and StemFit medium present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the third culture medium does not contain antibiotics; j) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days; k) Remove the third culture medium from the tissue culture dish including the vertical wheel and add the wash medium at a rate of about 30 mL / min; l) Using a 40µm cell retention filter, replace approximately 3L to approximately 3.5L of the wash medium in the tissue culture dish including the vertical wheel at a pump flow rate of 30mL / min and remove 0.1L of the wash medium from the tissue culture dish including the vertical wheel; m) Add approximately 50 mL of wash medium and approximately 50 mL of a fourth medium to the tissue culture dish including the vertical wheel, wherein the fourth medium contains advanced DMEM / F12 medium and StemFit in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fourth culture medium does not contain antibiotics; n) The cells are cultured in the tissue culture dish including a vertical wheel and stirred at about 10 rpm to 60 rpm, preferably 23 rpm, for about 1.8 days to about 2.2 days, preferably about 2 days; o) Using a 4µm cell retention filter, at a pump flow rate of approximately 0.69 mL / min (1 L / day), simultaneously remove the fourth culture medium from the tissue culture dish including the vertical wheel and add the fifth culture medium to the tissue culture dish including the vertical wheel within approximately 1.8 days to approximately 2.2 days, preferably approximately 2 days, until 2 L of culture medium has been replaced. The fifth culture medium contains advanced DMEM / F12 medium and StemFit at a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® Differentiation medium, and also containing SCF, IL-6, TPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, in which the fifth culture medium does not contain antibiotics; p) Remove the entire cell volume containing EB and iHSC from the tissue culture dish including the vertical wheel and centrifuge the entire cell volume to produce a first supernatant and a first cell pellet; q) Centrifuge the first supernatant to produce a second supernatant and a second cell pellet. Remove the second supernatant and resuspend the second cell pellet in advanced DMEM / F12 medium and StemFit medium free of cytokines, growth factors, or antibiotics. ® In the differentiation culture medium; r) Add a first volume of 20 mg / mL collagenase II to the first cell pellet, resuspend the first cell pellet, and add a second volume of 20 mg / mL collagenase II to produce a first cell suspension. s) Add an equal volume of advanced DMEM / F12 medium and StemFit culture medium (free of cytokines, growth factors, or antibiotics) to the first cell suspension. ® Differentiation culture medium; t) The cell suspension is centrifuged to produce a third cell precipitate; u) Resuspend the third cell pellet in a certain volume of TrypLE and add a certain volume of advanced DMEM / F12 medium and StemFit medium that do not contain cytokines, growth factors or antibiotics. ® Differentiation culture medium, thereby producing a second cell suspension; v) Mix the second cell suspension until the cell clusters are no longer visible; w) If cell clusters are visible after the mixing step, the second cell suspension may optionally be passed through a cell filter to obtain iHSC, wherein the cell filter has a 70 µm mesh size; x) Centrifuge the second cell suspension; y) No serum is introduced in any of the steps a)-x); z) No additional cells, including feeder cells or matrix cells, are introduced in any of the steps a)-y). aa) Determine the purity of the iHSC from step w), the purity being defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2, and CD235a; and bb) Obtain the iHSC generated from scratch.

77. A method for de novo generation of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) from γδ T cell-derived iPSCs (γδ T-iPSCs) under serum-free, feeder-free culture conditions, said method comprising one or more of the following steps: i) Obtaining one or more human induced pluripotent stem cells (iPSCs), and said method further comprising one or more of the following steps: a) Inoculating the iPSCs, wherein the inoculation is performed by suspending the iPSCs in a first culture medium in a bioreactor to generate a first cell suspension, wherein the bioreactor is a vertical wheel bioreactor, and wherein the first culture medium contains StemFit. ® Basic 04 medium and StemFit ® One of the following: Basic 03 medium, CHIR99021 (glycogen synthase kinase inhibitor), bone morphogenetic protein 4 (BMP4), recombinant human vascular endothelial growth factor (rhVEGF), and one of the following: Rho kinase inhibitor (ROCK inhibitor) and a mixture of chromogen 1, enricane, polyamine and trans-ISRIB (CEPT). Furthermore, the first culture medium does not contain antibiotics; b) The first cell suspension is cultured and stirred in the bioreactor, wherein the first cell suspension is cultured and stirred for about 1.8 days to about 2.2 days, preferably about 2 days, wherein embryoid bodies (EB) are generated. c) Centrifuge the first cell suspension to produce a first cell precipitate; d) Resuspend the first cell pellet in a second culture medium to form a second cell suspension, wherein the second culture medium comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SB431542 (an inhibitor of the activin / BMP / TGFβ pathway), rhVEGF, basic fibroblast growth factor (bFGF), stem cell factor (SCF), and L-glutamine and GlutaMax. ™ One of the supplements, or ii) StemPro with a 500:13 ratio ™ -34 serum-free medium (SFM) and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, insulin-transferrin-selenium (ITS-G), monothioglycerol, SB431542, rhVEGF, bFGF, SCF, as well as L-glutamine and GlutaMax ™ One of them, Furthermore, the second culture medium does not contain antibiotics; e) The second cell suspension is cultured and stirred in the bioreactor for about 1.8 days to about 2.2 days, preferably about 2 days; f) Stop stirring for about 5 minutes, during which the cell aggregates settle in the second cell suspension; g) Collect approximately 80% of the top volume of the second cell suspension from the bioreactor; h) Centrifuge the collected second cell suspension to form a second cell pellet, and resuspend the second cell pellet in approximately 1 mL of StemFit solution. ® Differentiation solution and StemPro ™ -34 SFM in one of them; i) will be in approximately 1 mL of StemFit ® Differentiation solution and / or StemPro ™ The second cell pellet in one of the -34 SFMs is suspended in a third culture medium to form a third cell suspension, wherein the third culture medium comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains SCF, rhVEGF, FMS-like tyrosine kinase 3 ligand (Flt3L), interleukin-3 (IL-3), interleukin-6 (IL-6), one of (i) thrombopoietin (TPO) and (ii) erythropoietin (EPO), and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, rhVEGF, IL-3, IL-6, Flt3L, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them, Furthermore, the third culture medium does not contain antibiotics; j) The third cell suspension is placed into a bioreactor containing the remaining second cell suspension to form a fourth cell suspension; k) The fourth cell suspension is cultured and stirred in the bioreactor for about 1.8 days to about 2.2 days, preferably about 2 days; l) Stop stirring for about 5 minutes; during which time the cell aggregates settle in the cell suspension; m) Collect approximately 80% of the fourth cell suspension and centrifuge the collected fourth cell suspension to form a third cell pellet, and resuspend the third cell pellet in approximately 1 mL of StemFit. ® Differentiation solution and StemPro ™ -34 SFM in one of them; n) Dissolve in approximately 1 mL of StemFit ® Differentiation solution and StemPro ™ The third cell pellet in one of the -34 SFMs is suspended in a fourth culture medium to form a fifth cell suspension, wherein the fourth culture medium comprises: i) Advanced DMEM / F12 medium and StemFit present in a ratio of approximately 3.9:0.9 to 4.1:1.1, preferably 4:

1. ® The differentiation medium also contains one of SCF, IL-6, (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax. ™ One of them, or ii) StemPro with a 500:13 ratio ™ -34 SFM and StemPro ™ -34 nutritional supplements, ascorbic acid 2-phosphate, ITS-G, monothioglycerol, SCF, IL-6, one of (i) TPO and (ii) EPO, and (i) L-glutamine and (ii) GlutaMax ™ One of them, Furthermore, the fourth culture medium does not contain antibiotics; o) The fifth cell suspension is placed into the bioreactor containing the remaining fourth cell suspension to form a sixth cell suspension; p) The sixth cell suspension is cultured and stirred in the bioreactor for about 1.8 days to about 2.2 days, preferably about 2 days; q) Repeat steps l) to p); r) Remove all of the eighth cell suspension from the bioreactor and harvest EB and iHSC from the cell suspension by centrifugation, thereby producing a fourth cell precipitate and a first supernatant; s) Collect the first supernatant; t) The first supernatant is centrifuged to produce a fifth cell pellet, and the fifth cell pellet is resuspended in a fifth culture medium to produce a ninth cell suspension, wherein the fifth culture medium comprises advanced DMEM / F12 medium, wherein the fifth culture medium does not contain cytokines, antibiotics or growth factors, and wherein the cells of the ninth cell suspension comprise iHSCs defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a; u) The fourth cell pellet from step r) is resuspended in a certain volume of about 20 mg / mL type II collagenase to produce the tenth cell suspension; v) Add the fifth culture medium to the tenth cell suspension; w) Centrifuge the tenth cell suspension to produce a sixth cell precipitate, and add TrypLE to the sixth cell precipitate to produce an eleventh cell suspension. x) Add the additional fifth culture medium to the eleventh cell suspension and mix; y) The eleventh cell suspension is centrifuged to form a seventh cell pellet, and the seventh cell pellet is resuspended in another fifth culture medium to form a twelfth cell suspension; z) Pass the twelfth cell suspension through a cell filter to obtain iHSC, wherein the cell filter has a 70µm mesh size; aa) Determine the purity of the iHSC from the first supernatant of step t) and the iHSC from step z), wherein the purity is defined as CD34+ and negative for lineage markers, wherein the lineage markers include one or more of CD3, CD19, CD14, CD11b, CD11c, CD56, CD20, CD16, CD2 and CD235a; bb) No serum is introduced in any of the steps a)-aa); cc) No additional cells, including feeder cells or matrix cells, are introduced in any of steps a)-bb); and dd) to obtain the iHSC generated from scratch.

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