Method for constructing thymus organoid and use thereof

Abstract

Disclosed in the present invention are a method for constructing a thymus organoid and the use thereof. The method enables the differentiation of pluripotent stem cells into a thymus organoid under suspension culture conditions by using small molecule compounds and small molecule proteins in combination. The method of the present invention can be used to efficiently obtain a thymus organoid having a structure and function more similar to those of a natural thymus.

Description

Construction methods and applications of thymus organoids Technical Field

[0001] This invention belongs to the field of organoid technology, specifically relating to the construction method and application of thymus organoids. Background Technology

[0002] The thymus is an important immune organ in the human body, serving as the site of T cell differentiation and maturation. Aging or damage to the thymus can easily lead to decreased immunity, autoimmune diseases, or even malignant tumors. Therefore, thymic disorders severely impact the body's immune function, disrupting immune homeostasis and causing autoimmune diseases. T cell deficiencies caused by thymic dysfunction make patients highly susceptible to infections, leading to malignant tumors and / or autoimmune diseases. Besides thymic degeneration due to aging, damage to thymic epithelial cells (TECs) can be caused by many external factors, including infection, radiation, immunosuppressant therapy, and graft-versus-host disease after hematopoietic stem cell transplantation.

[0003] Organoids can differentiate into various organ-specific cell types and reproduce some functions and spatial structures of corresponding organs. Their emergence has injected new momentum into biomedical research and clinical applications, showing broad application prospects in fields such as basic and clinical cancer research and regenerative medicine. Previously, organoids of the brain, intestine, liver, lung, kidney, tongue, and retina have been successfully cultured. However, progress in the field of thymus organoids has been slow. For many years, researchers have mainly explored the development process of thymic epithelial cells (TECs) and revealed the mechanisms of thymic injury and regeneration through in vitro two-dimensional (2D) culture systems. However, monolayer 2D culture systems cannot reproduce the unique three-dimensional (3D) epithelial reticular structure of the thymus, nor can they adequately provide the cytokines and growth factors from other supporting cell sources required for the directed differentiation of hematopoietic stem cells into functional T cells; there are no reports of technologies for constructing 3D thymus organoids in vitro using human pluripotent stem cells.

[0004] Currently, three major research bottlenecks in this field are: first, the positive and negative selection processes of the human thymus cannot be simulated in vitro; second, the in vitro preparation of allogeneic T cells cannot achieve complete HLA matching between allogeneic individuals, resulting in immune rejection; and third, the in vitro preparation of functional CD4-positive T cells is difficult. However, to date, there have been no reports of differentiating truly functional, especially in vivo, thymic organoids from human pluripotent stem cells.

[0005] Previously, only a few researchers isolated thymic epithelial cells (TEC) and thymic stromal cells (TIC) from natural human thymus tissue and then constructed tissue-engineered thymuses with various scaffold materials, such as collagen scaffolds [1, 2, 3], decellularized thymic scaffolds [4, 5], and tantalum scaffolds [6, 7], to obtain a reassembled thymus-like structure in vivo. However, due to the very limited availability of natural human thymus samples, thymuses assembled from scaffolds or natural thymic epithelial cells or thymic stromal cells cannot be mass-produced to meet research and clinical needs.

[0006] Currently, some scholars differentiate human embryonic stem cells (hESCs) into thymic epithelial progenitor cells (TEP)[8]. The entire differentiation process is carried out in two-dimensional (2D) cell culture. After differentiating into thymic epithelial progenitor cells in vitro, they are transplanted into nude mice and eventually mature into thymic epithelial cells.

[0007] However, this differentiation process does not take into account the natural thymus structure and cellular composition. The thymus is composed of a variety of thymic stromal cells, rather than a single thymic epithelial cell. In addition to thymic epithelial cells, the thymic stroma also includes fibroblasts, endothelial cells, etc., which are components of the thymus parenchyma and vascular structure and play an indispensable role in the development and function of thymic epithelial cells[9], and are also necessary to ensure the thymus functions. Some thymic endothelial cells are surrounded by pericytes, which are specialized contractile fibroblast-like cells that express smooth muscle actin (α-SMA)

[0010] . They promote the maturation of thymic epithelial cells through cell-to-cell interactions.

[0008] The disadvantages of the existing technologies mentioned above are: 1. In vitro, human pluripotent stem cells can only be differentiated into thymic epithelial progenitor cells. Then, the differentiated cells need to be transplanted into immunodeficient mice, where they can further mature into thymic epithelial cells in the in vivo environment; 2. The differentiation process uses a lot of expensive cell growth factors, resulting in high reagent costs; 3. The 2D plate differentiation method does not take into account the three-dimensional developmental niche of the natural thymus organ; 4. This method differentiates into a single thymic epithelial progenitor cell and does not take into account the necessity of other stromal cells that make up the thymus, such as mesenchymal cells, fibroblasts, and endothelial cells, for supporting the thymic epithelial cells and for their sustained cell signaling regulation to enable the thymus to function.

[0009] References

[0010] [1] Suematsu S, Watanabe T. Generation of a synthetic lymphoid tissue-like organoid in mice. Nat Biotechnol, 2004, 22(12): 1539-1545.

[0011] [2] H,Sousa AE.Repairing thymic function.Curr Opin Organ Transplant,2013,18(3):363-368.

[0012] [3]Gostynska N,Shankar Krishnakumar G,Campodoni E,et al.3D porous collagen scaffolds reinforced by glycation with ribose for tissue engineering application.Biomed Mater,2017,12(5):055002.

[0013] [4]Campinoti S,Gjinovci A,Ragazzini R,et al.Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.Nat Commun,2020,11(1):6372.

[0014] [5]Schmitt TM, JC.Induction of T cell development from hematopoietic progenitor cells by delta-like-1in vitro.Immunity,2002,17(6):749-756.

[0015] [6]Bobyn JD,Stackpool GJ,Hacking SA,et al.Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial.J Bone Joint Surg Br,1999,81(5):907-914.

[0016] [7]Marshall D, Bagley J, Le P, et al. T cell generation including positive and negative selection ex vivo in a three-dimensional matrix. J Hematother Stem Cell Res, 2003, 12(5):565-574.

[0017] [8] Parent AV, Russ HA, Khan IS, et al. Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development. Cell Stem Cell. 2013; 13(2): 219-229.

[0018] [9] Nitta T, Takayanagi H. Non-Epithelial Thymic Stromal Cells: Unsung Heroes in Thymus Organogenesis and T Cell Development. Front Immunol. 2021; 11: 620894.

[0019] Ushiki T, Takeda M. Three-dimensional ultrastructure of the perivascular space in the rat thymus. Arch Histol Cytol (1997) 60:89-99. Summary of the Invention

[0020] The purpose of this application is to provide a method for constructing thymus organoids that fully simulates the development of natural thymus, and the constructed thymus organoid tissue structure, cell composition and function are highly consistent with natural thymus.

[0021] A first aspect of the present invention provides a culture medium combination, the culture medium combination comprising a first culture medium and a second culture medium; the first culture medium comprising a first basal culture medium supplemented with Activin A, GSK-3 inhibitor and ROCK inhibitor; the second culture medium comprising a second basal culture medium supplemented with Activin A and B-27 additives.

[0022] In some embodiments of the present invention, the GSK-3 inhibitor is selected from at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021.

[0023] In some embodiments of the present invention, the GSK-3 inhibitor is CHIR99021.

[0024] In some embodiments of the present invention, the ROCK inhibitor is selected from at least one of Y-27632, Thiazovivin, Fasudil HCl, GSK429286A, RKI-1447 and Azaindole 1.

[0025] In some embodiments of the present invention, the ROCK inhibitor is Y-27632.

[0026] In some embodiments of the present invention, the concentration of Activin A added to the first basal culture medium is 80-120 ng / mL.

[0027] In some embodiments of the present invention, the concentration of CHIR99021 added to the first basal culture medium is 4-6 μM.

[0028] In some embodiments of the present invention, the concentration of Y-27632 added to the first basal culture medium is 8-12 μM.

[0029] In some embodiments of the present invention, the concentration of Activin A added to the second basal culture medium is 80-120 ng / mL.

[0030] In some embodiments of the present invention, the concentration of B-27 additive in the second basal culture medium is 0.5-1.5×.

[0031] In some embodiments of the present invention, the culture medium combination further includes a third culture medium; the third culture medium includes a third basal culture medium supplemented with a TGF-β signaling pathway inhibitor and a Notch receptor agonist.

[0032] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0033] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0034] In some embodiments of the present invention, the Notch receptor agonist is selected from DLL4.

[0035] In some embodiments of the present invention, the concentration of SB431542 added to the third basal culture medium is 8-12 μM.

[0036] In some embodiments of the present invention, the concentration of DLL4 added to the third basal culture medium is 8-12 ng / mL.

[0037] In some embodiments of the present invention, the culture medium combination further includes a fourth culture medium, which includes a fourth basal culture medium supplemented with a TGF-β signaling pathway inhibitor, a Notch receptor agonist, and cytokines.

[0038] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0039] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0040] In some embodiments of the present invention, the Notch receptor agonist includes DLL4.

[0041] In some embodiments of the present invention, cytokines include FLT3L, TPO, and SCF.

[0042] In some embodiments of the present invention, the concentration of SB431542 added to the fourth basal culture medium is 8-12 μM.

[0043] In some embodiments of the present invention, the concentration of DLL4 added to the fourth basal culture medium is 8-12 ng / mL.

[0044] In some embodiments of the present invention, the concentration of TPO added to the fourth basal culture medium is 4-6 ng / mL.

[0045] In some embodiments of the present invention, the concentration of FLT3L added to the fourth basal culture medium is 4-6 ng / mL.

[0046] In some embodiments of the present invention, the concentration of SCF added in the fourth basal culture medium is 40-60 ng / mL.

[0047] In some embodiments of the present invention, the first basal culture medium and the second basal culture medium are stem cell culture media;

[0048] The third and fourth basic culture media are endothelial cell culture media, which are selected from any one of EGM2 medium, ECM medium, and VascuLife VEGF Medium Complete Kit medium.

[0049] In some embodiments of the present invention, the first basal culture medium and the second basal culture medium are X-VIVO 20 culture medium.

[0050] In some embodiments of the present invention, the third basal culture medium and the fourth basal culture medium are EGM2 culture medium.

[0051] A second aspect of the present invention provides a kit comprising the culture medium combination provided in the first aspect of the present invention.

[0052] In some embodiments of the present invention, trophoblast cells are also included.

[0053] The third aspect of the present invention provides the application of the culture medium combination provided in the first aspect of the present invention or the kit provided in the second aspect of the present invention: (1) preparing mesoderm cells; (2) preparing third pharyngeal pouch endoderm cells; (3) preparing thymus organoids; (4) preparing products that induce pluripotent stem cells to differentiate into mesoderm cells; (5) preparing products that induce pluripotent stem cells to differentiate into third pharyngeal pouch endoderm cells; (6) preparing products that induce pluripotent stem cells to differentiate into thymus organoids.

[0054] The fourth aspect of the present invention provides a method for preparing mesoderm cells and / or third pharyngeal pouch endoderm cells and / or thymus organoids, wherein mesoderm cells and / or third pharyngeal pouch endoderm cells and / or thymus organoids are obtained by culturing pluripotent stem cells using a kit provided in the second aspect of the present invention; wherein the culture is a suspension culture.

[0055] In some embodiments of the present invention, the method specifically includes:

[0056] (1) Pluripotent stem cells are cultured sequentially in the first culture medium and the second culture medium of the kit provided in the second aspect of the present invention to obtain mesoderm cells; and / or

[0057] (2) Third pharyngeal pouch endoderm cells are obtained by co-culturing mesoderm cells and trophoblast cells in the third culture medium of the kit provided in the second aspect of the present invention; and / or

[0058] (3) Thymoid organoids were obtained by culturing the third pharyngeal endoderm cells in the fourth culture medium of the kit provided in the second aspect of the present invention.

[0059] In some embodiments of the present invention, the method specifically includes:

[0060] (1) Pluripotent stem cells were cultured sequentially in the first and second culture media in the kit to obtain mesoderm cells; and / or

[0061] (2) Third pharyngeal pouch endoderm cells were obtained by co-culturing mesoderm cells and trophoblast cells in the third culture medium provided in the kit; and / or

[0062] (3) Thymoid organoids were obtained by culturing the third pharyngeal pouch endoderm cells in the fourth culture medium in the kit.

[0063] The first culture medium includes a first basal culture medium supplemented with Activin A, GSK-3 inhibitor and ROCK inhibitor; the second culture medium includes a second basal culture medium supplemented with Activin A and B-27 additive; the third culture medium includes a third basal culture medium supplemented with TGF-β signaling pathway inhibitor and Notch receptor agonist; and the fourth culture medium includes a fourth basal culture medium supplemented with TGF-β signaling pathway inhibitor, Notch receptor agonist and cytokines.

[0064] The fifth aspect of this invention provides the use of thymus organoids prepared by the method provided in the fourth aspect of this invention in any of the following: 1) for constructing humanized blood and / or immune system animals; 2) for preparing products for constructing humanized blood and / or immune system animals; 3) for simulating human diseases; 4) for preparing products for simulating human diseases; 5) for preparing human T cells; 6) for preparing products for preparing human T cells; 7) for preparing products for inducing transplant tolerance; 8) for preparing products for restoring thymus function; 9) for generating humanized antibodies; 10) for generating humanized antibodies; 11) for screening, developing and / or analyzing the toxicity of thymus-related drugs; 12) for studying the pathogenesis of thymus diseases; 13) for preparing products for treating thymus diseases; 14) for constructing thymus disease models; 15) for thymus toxicology testing.

[0065] The beneficial effects of this invention are:

[0066] This invention provides a culture medium combination for inducing the directed differentiation of pluripotent stem cells into thymic organoids. By using a combination of small molecule compounds and small molecule proteins, pluripotent stem cells can be differentiated into thymic organoids, resulting in the efficient acquisition of thymic organoids with structures and functions more closely resembling those of the natural thymus. The entire differentiation process involves first differentiating pluripotent stem cells into mesoderm, then directionally inducing the formation of the third pharyngeal pouch endoderm, which subsequently differentiates into thymic organoids.

[0067] This invention also provides a method for inducing pluripotent stem cells to differentiate into thymus organoids. This invention establishes a highly efficient method for inducing pluripotent stem cell differentiation into thymus organoids through 3D suspension culture by combining different concentrations of small chemical molecules and small protein molecules. Compared with 2D adherent culture induction system, the 3D suspension culture differentiation system has the advantage of better simulating the three-dimensional microenvironment of the human body. Therefore, the 3D suspension culture differentiation system can efficiently obtain thymus organoids with a structure and function closer to the natural thymus. Attached Figure Description

[0068] Figure 1. Schematic diagram of the method for directed induction of human pluripotent stem cells into thymic organoids.

[0069] Figure 2 shows the relevant detection results of the mesoderm cell differentiation stage in Example 2. Among them, Figure 2A is the immunofluorescence staining of human pluripotent stem cell stem genes SOX2 and OCT4; Figure 2B is the flow cytometry detection of human pluripotent stem cell stem gene SSEA4; Figure 2C is the RT-qPCR detection of stem genes, ectoderm, mesoderm and endoderm marker genes during mesoderm differentiation; Figure 2D is the image of morphological changes of mesoderm cell spheres in directional induced differentiation, scale bar = 50 μm; Figure 2E is the immunofluorescence image of mesoderm cell spheres FoxA2 and BRA; Figure 2F is the flow cytometry detection image of mesoderm cell spheres CXCR4 and SOX17; Figure 2G is the flow cytometry detection image of mesoderm cell spheres co-stained with CXCR4 and SOX17.

[0070] Figures 3A-3B show the relevant detection results of the third pharyngeal pouch endoderm differentiation stage in Example 2. Figure 3A shows the RT-qPCR detection of key genes in the third pharyngeal pouch endoderm; Figure 3B shows the cell flow cytometry analysis of the key genes SIX1 and PAX9 in the third pharyngeal pouch endoderm.

[0071] Figure 4 shows the relevant detection results of the thymus organoid differentiation stage in Example 2. Figure 4A shows the RT-qPCR detection of key genes K5, K8, and EPCAM in thymus epithelial cells of thymus organoids; Figure 4B shows the flow cytometry analysis of thymus epithelial cells expressing stem CD90 and EPCAM in thymus organoids; Figure 4C shows the RT-qPCR and flow cytometry analysis of the transcription factor FOXN1 in thymus epithelial cells of thymus organoids; and Figure 4D shows the RT-qPCR and flow cytometry analysis of the autoimmune regulatory factor AIRE in thymus organoids. Detection: Figure 4E shows the RT-qPCR and flow cytometry detection of MHC-II in thymic organoids; Figure 4F shows the RT-qPCR detection of representative genes in thymic organoid stromal cells; Figure 4G shows the RT-qPCR detection of representative functional genes in thymic organoids; Figure 4H shows the immunofluorescence of key proteins K5 and K8 in thymic epithelial cells of thymic organoids, scale bar = 50 μm; Figure 4I shows the immunofluorescence staining of key transcription factor FOXN1 and mesenchymal cell marker PDGFRa in thymic epithelial cells of thymic organoids, scale bar = 50 μm.

[0072] Figure 5 shows the detection results of thymus organoids in Example 3. Figure 5A is the integrated UAMP diagram of natural thymus samples and thymus organoids at different time points; Figure 5B is the correlation analysis of natural thymus samples and thymus organoids at different time points; Figure 5C is the integrated UAMP diagram of thymus samples and thymus organoids at 19 weeks of gestation; and Figure 5D is a columnar stacking diagram of the proportion of cell groups in the thymus and thymus organoids of 19 weeks of gestation.

[0073] Figure 6 shows the relevant schematic diagrams and detection results of the co-culture system in Example 4. Figure 6A is a schematic diagram of the co-culture system of thymus organoids and hematopoietic stem / progenitor cells; Figure 6B shows the CD45 levels in the co-culture system at the indicated time points. + Representative dynamics of cell differentiation, DAPI - The gate shows the total number of live cells; Figure 6C shows the number of T progenitor cells (CD55) in the co-culture system at the time point shown. + CD7 + Representative dynamics of differentiation, DAPI - CD45 + The gate shows total live cells; Figure 6D shows mature T(CD3) cells in the co-culture system. + TCRαβ + A representative diagram of cell differentiation ratios; Figure 6E shows mature T(CD3) cells in the co-culture system. + TCRαβ + )Statistical graph of cell differentiation ratio; Figure 6F is the CD3 in the co-culture system + Microscopic images and immunofluorescence staining of T cells, a representative immunofluorescence image at week 6 of co-culture, green for CD3 staining; blue for DAPI (nuclear) staining, scale bar = 20 μm; Figure 6G shows the representative kinetics of differentiation of mature CD4SP and CD8SP T cells in the co-culture system at the indicated time point, DAPI... - CD45 + The gate displays the total number of live cells; Figure 6H shows the co-culture system at a specified time point. CD4 SP (CD3) + TCRαβ + CD45RA + CD45RO - CCR7 + CD4 + CD8 - ) and CD8 SP (CD3 + TCRαβ + CD45RA + CD45RO - CCR7 + CD4 - CD8 + Cell ratio statistics (for DAPI) - CD45 + (Cell gating); Figure 6I shows CD3 in the co-culture system at week 6. + TCRγδ + A representative diagram of T cell differentiation, with total live cells displayed in DAPI. - CD45 +In gating; Figure 6J shows CD3 in the co-culture system at week 6. + TCRγδ + T cell ratio statistics (gated in DAPI) - CD45 + (on cells); Figure 6K shows CD4 in the co-culture system. + CD25 + The proportion of T cells and CD4+ in the co-culture system at week 6 + CD25 + A representative diagram of T cell differentiation, with total live cells displayed in DAPI. - CD45 + CD3 + TCRαβ + Gated.

[0074] Figure 7 shows the relevant detection results in Example 5. Figure 7A shows the flow cytometry analysis of human CD45 in the peripheral blood of hETO group mice 8 weeks after HSPC transplantation. + CD3 + The proportion of T cells; Figure 7B shows the human CD45 concentration in the peripheral blood of hETO mice 12 weeks after HSPC transplantation, analyzed by flow cytometry. + CD3 + The proportion of T cells; Figure 7C shows the flow cytometry analysis of human CD45 in the peripheral blood of hETO group mice 16 weeks after HSPC transplantation. + CD3 + The proportion of T cells; Figure 7D shows the expression of CD45, CD8, CD4, CD3, TCRαβ, CD25, FOXP3 and TCRγδ in the bone marrow of hETO mice 11 months after HSPC transplantation from severely immunodeficient mice by flow cytometry; Figure 7E shows the expression of CD45, CD8, CD4, CD3, TCRαβ, CD25, FOXP3 and TCRγδ in the spleen of hETO mice 11 months after HSPC transplantation from severely immunodeficient mice by flow cytometry.

[0075] Figure 8 shows the relevant detection results in Example 6. Figure 8A shows the levels of human ovalbumin-specific IgM (left) and IgG (right) after one week, as shown by ELISA analysis; Figure 8B shows the levels of human ovalbumin-specific IgM (left) and IgG (right) after two weeks, as shown by ELISA analysis.

[0076] Figure 9 shows the relevant detection results in Example 7. Figure 9A shows the proportion of human T cells after allogeneic skin transplantation as detected by flow cytometry. Figure 9B is an HE-stained image of the transplanted skin graft collected on day 6, scale bar = 300 μm. Figure 9C shows the expression of human CD3 (green) and IL-10 (red) in the transplanted skin graft using immunofluorescence staining, with cell nuclei stained with DAPI, scale bar = 20 μm. Figure 9D shows the peripheral blood CD3 expression in recipient mice on day 4 as detected by flow cytometry. + T cells express human cytokines IFN-γ, TNFα, and IL-2.

[0077] Figure 10 shows the relative expression levels of germ layer and mesoderm marker gene mRNAs detected by qRT-PCR from day 0 to day 3 of differentiation, where A: Activin A; A+B+W: Activin A, B-27 additive, Wnt3A; A+B+C: Activin A, B-27 additive, and CHIR99021.

[0078] Figure 11 shows the relative expression levels of thymic marker gene mRNAs on day 17 of differentiation as detected by qRT-PCR, where A: Activin A; A+B+W: Activin A, B-27 additive, Wnt3A; A+B+C: Activin A, B-27 additive, and CHIR99021. Detailed Implementation

[0079] In a first aspect, the present invention provides a culture medium combination comprising a first culture medium and a second culture medium; the first culture medium comprising a first basal culture medium supplemented with Activin A, a GSK-3 inhibitor and a ROCK inhibitor; and the second culture medium comprising a second basal culture medium supplemented with Activin A and B-27 additives.

[0080] In this invention, the first basal culture medium and the second basal culture medium refer to commercially available culture media suitable for stem cell culture. The "first basal culture medium with added Activin A, GSK-3 inhibitor and ROCK inhibitor" is obtained by adding Activin A, GSK-3 inhibitor and ROCK inhibitor to a commercially available culture medium suitable for stem cell culture. The "second basal culture medium with added Activin A and B-27 additive" is obtained by adding Activin A and B-27 additive to a commercially available culture medium suitable for stem cell culture.

[0081] In some embodiments of the present invention, the GSK-3 inhibitor is selected from at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021.

[0082] In some embodiments of the present invention, the GSK-3 inhibitor is CHIR99021. CHIR99021 in the present invention includes CHIR99021 and its salts, particularly pharmaceutically acceptable salts.

[0083] In some embodiments of the present invention, the ROCK inhibitor is selected from at least one of Y-27632, Thiazovivin, Fasudil HCl, GSK429286A, RKI-1447 and Azaindole 1.

[0084] In some embodiments of the present invention, the ROCK inhibitor is Y-27632. Y-27632 in the present invention includes Y-27632 and its salts, particularly pharmaceutically acceptable salts.

[0085] In some embodiments of the present invention, the concentration of Activin A added to the first basal culture medium is 80-120 ng / mL. Here, the concentration = mass of added Activin A / volume of the first basal culture medium.

[0086] In some embodiments of the present invention, the concentration of CHIR99021 added to the first basal culture medium is 4-6 μM. Here, the concentration = the amount of CHIR99021 added / the volume of the first basal culture medium.

[0087] In some embodiments of the present invention, the concentration of Y-27632 added to the first basal culture medium is 8-12 μM. Here, the concentration = the amount of Y-27632 added / the volume of the first basal culture medium.

[0088] In some embodiments of the present invention, the concentration of Activin A added to the second basal culture medium is 80-120 ng / mL. Here, the concentration = mass of added Activin A / volume of the second basal culture medium.

[0089] In some embodiments of the present invention, the concentration of B-27 additive in the second basal culture medium is 0.5-1.5×. Here, the concentration = multiple of the original B-27 additive solution * (volume of added B-27 additive / volume of the second basal culture medium). For example, if the B-27 additive is provided in 50× liquid form, the original multiple is 50×.

[0090] In some embodiments of the present invention, the culture medium combination further includes a third culture medium; the third culture medium includes a third basal culture medium supplemented with a TGF-β signaling pathway inhibitor and a Notch receptor agonist.

[0091] In this invention, the third basal culture medium refers to a commercially available culture medium that can be used for endothelial cell culture. The "third basal culture medium with added TGF-β signaling pathway inhibitors and Notch receptor agonists" is obtained by adding TGF-β signaling pathway inhibitors and Notch receptor agonists to a commercially available culture medium that can be used for endothelial cell culture.

[0092] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0093] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0094] In some embodiments of the present invention, the Notch receptor agonist is selected from DLL4.

[0095] In some embodiments of the present invention, the concentration of SB431542 added to the third basal culture medium is 8-12 μM. Here, the concentration = the amount of SB431542 added / the volume of the third basal culture medium.

[0096] In some embodiments of the present invention, the concentration of DLL4 added to the third basal culture medium is 8-12 ng / mL. Here, the concentration = mass of added DLL4 / volume of the third basal culture medium.

[0097] In some embodiments of the present invention, the fourth culture medium includes a fourth basal culture medium supplemented with TGF-β signaling pathway inhibitors, Notch receptor agonists, and cytokines.

[0098] In this invention, the fourth basic culture medium refers to a commercially available culture medium that can be used for endothelial cell culture. The "fourth basic culture medium with added TGF-β signaling pathway inhibitors, Notch receptor agonists, and cytokines" is obtained by adding TGF-β signaling pathway inhibitors, Notch receptor agonists, and cytokines to a commercially available culture medium that can be used for endothelial cell culture.

[0099] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761.

[0100] In some embodiments of the present invention, the TGF-β signaling pathway inhibitor is SB431542.

[0101] In some embodiments of the present invention, the Notch receptor agonist includes DLL4.

[0102] In some embodiments of the present invention, cytokines include FLT3L, TPO, and SCF.

[0103] In some embodiments of the present invention, the concentration of SB431542 added to the fourth basal culture medium is 8-12 μM. Here, the concentration = the amount of SB431542 added / the volume of the fourth basal culture medium.

[0104] In some embodiments of the present invention, the concentration of DLL4 added to the fourth basal culture medium is 8-12 ng / mL. Here, the concentration = mass of added DLL4 / volume of the fourth basal culture medium.

[0105] In some embodiments of the present invention, the concentration of TPO added to the fourth basal medium is 4-6 ng / mL. Here, the concentration = mass of added TPO / volume of the fourth basal medium.

[0106] In some embodiments of the present invention, the concentration of FLT3L added to the fourth basal medium is 4-6 ng / mL. Here, the concentration = mass of added FLT3L / volume of the fourth basal medium.

[0107] In some embodiments of the present invention, the concentration of SCF added to the fourth basal culture medium is 40-60 ng / mL. Here, the concentration = mass of added SCF / volume of the fourth basal culture medium.

[0108] In some embodiments of the present invention, the first basal culture medium and the second basal culture medium are X-VIVO 20 culture medium.

[0109] In some embodiments of the present invention, the third and fourth basal culture media are EGM2 culture media.

[0110] A second aspect of the present invention provides a kit comprising the culture medium combination provided in the first aspect of the present invention.

[0111] In some embodiments of the present invention, the kit includes a first culture medium and a second culture medium of the first aspect of the present invention; used to induce pluripotent stem cells to differentiate into mesoendothelial cells.

[0112] "Pluripotent stem cells" refer to stem cells that possess the pluripotency to differentiate into all cells present in an organism, namely the three germ layers (endoderm, mesoderm, and ectoderm), and also have the ability to proliferate. There are no particular limitations on the aforementioned pluripotent stem cells; examples include embryonic stem cells (ES cells), embryonic stem cells (ntES cells) derived from cloned embryos obtained through nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. Pluripotent stem cells are preferably selected from at least one type selected from the group consisting of ES cells, ntES cells, and iPS cells. In one aspect of the present invention, pluripotent stem cells are preferably iPS cells or ES cells. The organism from which the pluripotent stem cells are derived is not particularly limited, but pluripotent stem cells derived from mammals are preferred, and pluripotent stem cells derived from primates are more preferred. Specifically, pluripotent stem cells derived from humans are an example.

[0113] The human embryonic stem cells in this invention are not human embryonic stem cells isolated or obtained from human embryos that have developed in vivo, nor are they stem cells isolated or obtained from human embryos that have not developed in vivo and have been fertilized for more than 14 days.

[0114] In some embodiments of the present invention, the kit further comprises a third culture medium for inducing mesoendodermal cells to differentiate into third pharyngeal endodermal cells.

[0115] In some embodiments of the present invention, the kit further comprises a fourth culture medium and feeder cells for inducing the differentiation of third pharyngeal endoderm cells into thymus organoids.

[0116] In some embodiments of the present invention, the trophoblast cells include at least one of OP9 cells, MS5 cells, HS-5 cells, or other fibroblasts.

[0117] In a third aspect of the present invention, the application of the culture medium combination provided in the first aspect of the present invention or the kit provided in the second aspect of the present invention is provided for: (1) preparing mesoendodermal cells; (2) preparing third pharyngeal pouch endoderm cells; (3) preparing thymus organoids; (4) preparing products for inducing pluripotent stem cells to differentiate into mesoendodermal cells; (5) preparing products for inducing pluripotent stem cells to differentiate into third pharyngeal pouch endoderm cells; and (6) preparing products for inducing pluripotent stem cells to differentiate into thymus organoids.

[0118] In a fourth aspect, the present invention provides a method for preparing mesoendodermal cells and / or third pharyngeal pouch endoderm cells and / or thymus organoids, wherein mesoendodermal cells and / or third pharyngeal pouch endoderm cells and / or thymus organoids are obtained by culturing using a kit provided in the second aspect of the present invention.

[0119] In some embodiments of the present invention, the culture is a suspension culture.

[0120] In some embodiments of the present invention, the method specifically includes:

[0121] (1) Pluripotent stem cells were cultured successively in the first and second culture media of the kit provided in the second aspect to obtain mesoendodermal cells; and / or

[0122] (2) Third pharyngeal pouch endoderm cells were obtained by co-culturing mesoendodermal cells and trophoblast cells in the third culture medium of the kit provided in the second aspect; and / or

[0123] (3) Thymoid organoids were obtained by culturing the third pharyngeal endoderm cells in the fourth culture medium provided in the kit in the second aspect.

[0124] In some embodiments of the present invention, the culture time for inducing pluripotent stem cells to differentiate into mesoendothelial cells is 2-4 days.

[0125] In some embodiments of the present invention, the culture time using the first culture medium is 0.5-1.5 days.

[0126] In some embodiments of the present invention, the culture time using the second culture medium is 1.5-2.5 days.

[0127] In some embodiments of the present invention, the culture time for inducing mesoderm cells to differentiate into third pharyngeal bud endoderm cells is 6-8 days.

[0128] In some embodiments of the present invention, the culture time for inducing the differentiation of third pharyngeal pouch endoderm cells into thymic organoids is 6-10 days.

[0129] In a fifth aspect, the invention provides the use of thymus organoids prepared by the preparation method provided in the fourth aspect of the invention in any of the following: 1) for constructing humanized blood and / or immune system animals; 2) for preparing products for constructing humanized blood and / or immune system animals; 3) for simulating human diseases; 4) for preparing products for simulating human diseases; 5) for preparing human T cells; 6) for preparing products for preparing human T cells; 7) for preparing products for inducing transplant tolerance; 8) for preparing products for restoring thymus function; 9) for generating humanized antibodies; 10) for generating humanized antibodies; 11) for screening, developing and / or analyzing the toxicity of thymus-related drugs; 12) for studying the pathogenesis of thymus diseases; 13) for preparing products for treating thymus diseases; 14) for constructing thymus disease models; 15) for thymus toxicology testing.

[0130] The thymus organoids prepared by this invention can have multiple uses, for example:

[0131] 1) Establishment of humanized blood and immune system animals

[0132] Currently, humanized mouse models of the blood and immune system are created by implanting human hematopoietic stem cells and natural thymus tissue into immunodeficient mice, thereby reconstructing the human blood and immune system. These models effectively reconstruct the human immune system and better mimic human immune characteristics. However, natural human thymus resources are scarce and cannot meet the needs of humanized animals. This invention can directly induce human pluripotent stem cells to produce an unlimited supply of thymus organoids, which can meet the needs of preparing humanized immune system model animals.

[0133] 2) Research on immunity and tumor immunity based on humanized immune system animal models

[0134] Wild-type mice exhibit a strong rejection response to xenogeneic cells and tissues derived from humans. While human tumor transplantation models relying on immunodeficient mice have been widely used in tumor immunology and novel therapy development, the lack of a human immune system and tumor immune microenvironment significantly limits translational research into immune mechanisms and immunotherapies. Therefore, the humanized immune system animal model of this invention can realistically simulate the interaction between human tumor cells and the immune system, which is of great significance for tumor immunology research, the development of human diseases, and novel therapies.

[0135] 3) Virological research based on animal disease models with humanized immune systems

[0136] In the field of pathogenic microorganisms, especially in virus research, humanized mice are widely used, such as for HIV, EBV, dengue virus, HCV, and the novel coronavirus. Humanized mice can be used for drug testing of human inflammatory responses, evaluating the efficacy or toxicity of drugs in mouse models. However, the immune system of ordinary mice remains rodent-derived; this genus difference prevents the replication of human viral infections in ordinary mice, failing to truly simulate the actual human condition. While primates can accurately simulate most human viral infections, their high cost and complex operation limit their widespread application. Therefore, the humanized mouse model of the immune system constructed in this invention can be widely used in human virological research.

[0137] 4) In vitro preparation of CD8-positive T immune cells suitable for clinical treatment

[0138] Human T lymphocytes must undergo thymic conditioning during development or differentiation to undergo negative selection, screening out T lymphocytes that have an immune response to the human body. This invention constructs a human thymus organoid and co-cultures it with human hematopoietic stem cells in vitro. The human T lymphocytes differentiated from the human hematopoietic stem cells are then conditioned to produce CD8-positive T immune cells that can be used clinically in vitro, avoiding the risk of transplanted T cells causing autoimmune diseases in patients.

[0139] 5) In vitro preparation of CD4-positive T immune cells and immunomodulatory cells suitable for clinical treatment.

[0140] Currently, there are technical challenges in preparing CD4-positive T immune cells in vitro. This invention, by co-culturing human thymus organoids with human hematopoietic stem cells, results in a significantly higher proportion of CD4-positive T immune cells differentiated from the human hematopoietic stem cells compared to CD8-positive T immune cells. Furthermore, CD4-positive T cells prepared from the co-culture of human hematopoietic stem cells and human thymus organoids can be induced into immunomodulatory cells, or they can be directly differentiated into immunomodulatory cells. Therefore, it is possible to produce clinically applicable CD4-positive T immune cells and immunomodulatory cells in vitro.

[0141] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0142] Example 1: A kit for directed induction of thymus organoid differentiation

[0143] A kit comprising a first culture medium, a second culture medium, a third culture medium, and a fourth culture medium.

[0144] The first culture medium was X-VIVO 20 medium (purchased from Lonza) with 100 ng / mL Activin A (purchased from Peprotech), 5 μM CHIR99021 (purchased from Selleckchem), and 10 μM Y-27632 (purchased from Selleckchem).

[0145] The second culture medium was: X-VIVO 20 medium with 1×B-27 additive (purchased from Gibco) and 100 ng / mL Activin A added;

[0146] The third culture medium was: EGM2 medium (purchased from Lonza) containing 10 uM SB431542 (purchased from MCE) and 10 ng / mL DLL4 (purchased from Peprotech);

[0147] The fourth culture medium was: EGM2 medium containing 10 uM SB431542, 10 ng / mL DLL4, 5 ng / mL TPO (purchased from Peprotech), 5 ng / mL FLT3L (purchased from Peprotech), and 50 ng / mL SCF (purchased from Peprotech).

[0148] The kit also includes OP9 bone marrow stromal cells (purchased from the Cell Bank of the Chinese Academy of Sciences).

[0149] Example 2: A method for directional induction of thymus organoid differentiation from human pluripotent stem cells (hPSCs) in 3D suspension culture.

[0150] This embodiment provides a method for differentiating pluripotent stem cells into thymic organoids: the differentiation of thymic organoids requires sequential induction of the mesoderm and the third pharyngeal pouch endoderm, which eventually develop into thymic organoids (Figure 1).

[0151] The differentiation process employed 3D suspension culture, with pluripotent stem cells cultured for 3 days (days 0-3 in Figure 1) using Activin A, CHIR99021, Y-27632, and 1×B-27 additives to complete the mesoderm differentiation stage. Subsequently, under the influence of DLL4 and SB431542, the cells underwent a 7-day third pharyngeal pouch endoderm differentiation stage (days 3-10 in Figure 1), after which SCF, TPO, and FLT3L cytokines were added to the culture medium. Harvesting occurred 7 days later (days 10-17 in Figure 1).

[0152] Compared with existing methods, the differences are as follows: First, it uses the small molecule CHIR99021 to replace the expensive growth factor Wnt3a for mesoendodermal cell differentiation and uses fewer cytokine combinations to differentiate thymic organoids; second, the use of 3D suspension culture can better simulate the developmental niche of natural thymic organoids; third, it does not require scaffolds or other auxiliary materials, as the thymic organoids can generate their own extracellular matrix to provide support; fourth, thymic organoids can be differentiated from a single human pluripotent stem cell to simultaneously produce thymic epithelial cells that perform thymic function, as well as all supporting cells such as mesenchymal cells, fibroblasts, endothelial cells, and pericytes, perfectly simulating the development process, composition, and structure of natural thymic cells, forming truly functional thymic organoids; both in vitro and in vivo, it can promote the development of human hematopoietic stem and progenitor cells into various functional T cells.

[0153] The specific steps are as follows:

[0154] 1. Mesoendodermal cell differentiation stage:

[0155] S1. Mesoendodermal cell differentiation stage

[0156] hESCs (obtained from WiCell) were digested in cell spheroids. After washing the cells with PBS, 1 mL of Gentle Cell Dissociation Reagent (GCDR, STEM CELL TECHNOLOGIES) was added, and the cells were incubated in a 37°C CO2 incubator for 3 minutes. Then, the GCDR in the wells was aspirated, and 1 mL of mesoderm differentiation medium-1 (first medium) was added. The cells were gently pipetted and collected into centrifuge tubes. After adding a certain volume of mesoderm differentiation medium-1, the cell suspension was evenly seeded into low-adhesion six-well plates and cultured overnight. On the second day, the cell spheroids were washed three times with PBS, and fresh mesoderm differentiation medium-2 (second medium) was added. The same medium was changed once daily. After a total of 3 days of treatment, human pluripotent stem cells were induced to differentiate into mesoderm cell spheroids.

[0157] The specific procedure for cell medium exchange is as follows: collect cell spheres into centrifuge tubes, use natural sedimentation or low-speed centrifugation to sink the cell spheres to the bottom of the tube, discard the supernatant, add 1 mL of PBS to wash the organoids, discard the supernatant, and then add the differentiation medium at the corresponding time point.

[0158] S2. Cell Harvest

[0159] On day 3 of culture, mesoendothelial cell spheres were analyzed by flow cytometry, immunofluorescence, and RT-qPCR. Mesoendothelial cell spheres were collected, allowed to settle naturally, and after discarding the supernatant, 1 mL of PBS was added to wash the spheres. Then, 1 mL of TrypLE was added.TM Digestion with Express enzyme at room temperature or fixation with 4% paraformaldehyde.

[0160] In the first stage (days 0-3), the differentiation system promotes the formation of endoderm containing a small portion of mesoderm, which then transitions to the third pharyngeal pouch endoderm and finally differentiates into thymus organoids.

[0161] Prior to differentiation, the pluripotency of day 0 hPSCs was assessed by immunofluorescence and flow cytometry. The results showed that most cells co-expressed SOX2 and OCT4 (Figure 2A), and SSEA4... + Cells exceeding 96% of the total cells (Fig. 2B); cells that have undergone the mesoendodermal cell differentiation stage showed gradual downregulation of pluripotency marker genes (OCT4 and NANOG) and neuroectodermal marker genes (SOX1), while endoderm-related genes (FOXA2, SOX17, CXCR4, and GATA6) were rapidly upregulated; mesodermal (BRA, Brachyury) genes were initially significantly upregulated, followed by rapid downregulation (Fig. 2C).

[0162] HE staining was performed on mesoendodermal cell spheres (Figure 2D). Immunofluorescence on day 3 showed that most cells expressed the endoderm marker FOXA2, while a small number of cells expressed the mesoendodermal marker Brachyury (BRA) (Figure 2E); cell flow cytometry analysis further confirmed the expression of the key endoderm gene CXCR4. + The cell percentage was 93.8%, SOX17 + The cell proportion was 51.3%, CXCR4 + SOX17 + The cell percentage was 50.2% (Fig. 2F, Fig. 2G).

[0163] These results indicate that this 3D differentiation can successfully differentiate hPSCs into endoderm, accompanied by the formation of a small amount of mesoderm.

[0164] 2. Differentiation stage of the third pharyngeal pouch endoderm:

[0165] S1. Stage of endoderm differentiation in the third pharyngeal pouch

[0166] Collect the mesoendothelial cell spheres prepared in step 1 into centrifuge tubes, allow the cell spheres to settle to the bottom of the tubes by natural sedimentation, discard the supernatant, and add 1 mL of PBS to wash the cells.

[0167] Before the experiment, OP9 cells were revived, treated with mitomycin C, discarded, and washed five times with PBS. TrypLE was used. TM OP9 cells were digested using Express Enzyme. 1.2 × 10⁻⁶ cells were then digested. 4OP9 cells were added to the third pharyngeal sac endoderm differentiation medium (third medium) and mixed with washed mesoderm cell spheres, and then added to low adhesion plates for culture.

[0168] S2. Cell Harvest

[0169] On day 10 of culture, flow cytometry, immunofluorescence, and RT-qPCR were performed on the endoderm cells from the third pharyngeal pouch. Cell spheres were collected, allowed to settle naturally, and the supernatant was discarded. The spheres were then washed with 1 mL of PBS. Subsequently, 1 mL of TrypLE was added. TM Digestion with Express enzyme at room temperature or fixation with 4% paraformaldehyde.

[0170] RT-qPCR results revealed that key markers of the third pharyngeal pouch endoderm (PAX8, FGF10, SIX1, ETV5, FGF3, TBX1, PAX9, DUSP6, PDX1) were expressed at the highest levels throughout the differentiation process (Figure 3A). Flow cytometry analysis performed on day 10 also showed that most cells expressed SIX1 and PAX9 (Figure 3B).

[0171] 3. Thymus organoid differentiation stage:

[0172] For the third stage, based on in vivo developmental cues, SCF, TPO and FLT3L were added sequentially to the culture medium from the second stage to induce thymus organoids.

[0173] S1. Cell exchange medium

[0174] Collect the third pharyngeal pouch endoderm cell spheres cultured and differentiated in step 2 into centrifuge tubes. Allow the cell spheres to settle to the bottom of the tubes by natural sedimentation. Discard the supernatant, add 5 mL of PBS to wash the cells, and discard the supernatant. From day 10 to day 17, add thymus organoid culture medium (fourth medium) to carry out thymus organoid differentiation. Change the culture medium once a day.

[0175] S2. Cell Harvest

[0176] On day 17 of culture, thymus organoids were analyzed by flow cytometry, immunofluorescence, and RT-qPCR. Cell spheres were collected, allowed to settle naturally, and the supernatant was discarded. The spheres were then washed with 1 mL of PBS. Subsequently, 1 mL of TrypLE was added. TM Digestion with Express enzyme at room temperature or fixation with 4% paraformaldehyde.

[0177] RT-qPCR was performed to detect the relative expression levels of marker genes in thymic epithelial cells (AIRE, Foxn1, K5, K8, EpCAM, MHC-II), mesenchymal cells (PDGFRA), endothelial cells (PECAM1), pericytes (PDGFRB), and vascular smooth muscle cells (ACTA2). The results showed that, with the progression of differentiation time, on day 17 of hETO development, the expression levels of all of the above genes were significantly higher than those in undifferentiated cells (day 0) (Figures 4A, 4C-4F) (p<0.05).

[0178] Furthermore, these cells possess characteristics of the thymus, FOXN1 + Cells (Fig. 4C), Aire + Cells (Fig. 4D), MHCII + The percentages of cells (Fig. 4E) were 56.5%, 59.7%, and 58.8%, respectively. Flow cytometry analysis confirmed CD90 expression in EpCAM-positive cells (Fig. 4B). Thymus function-related genes DLL4 and CCL25 were gradually upregulated during hETO development (Fig. 4G).

[0179] More importantly, immunofluorescence staining revealed the expression of markers for various thymic stroma cell types in hETO. On the one hand, hETO regionally expressed the thymic cortical epithelial marker K8 and the thymic medullary epithelial marker K5 (Fig. 4H). On the other hand, hETO highly expressed the key thymic epithelial cell transcription factor FOXN1 and the mesenchymal cell marker PDGFRA (Fig. 4I). Therefore, day 17 of differentiation was identified as the hETO developmental stage, at which time multidimensional cell types matured and fused.

[0180] Example 3: Detection of thymus organoids

[0181] This embodiment first integrates and analyzes the single-cell transcriptome sequencing data obtained from the thymus organoids cultured in Example 2 with a large number of published human thymus transcriptomes from individuals with developmental ages between 19 weeks of gestation and 6 years of age after birth.

[0182] The results showed that thymic organoids were present in cell clusters in all subgroups of the natural thymus (Figure 5A). Correlation analysis of single-cell sequencing data from these samples using the Pearson parameter correlation test revealed the strongest correlation between thymic organoids and the natural thymus at 19 weeks of fetal development (Figure 5B).

[0183] Anchors were identified using the Find Integration Anchors function, and these anchors were used to integrate thymic organoids and natural thymic fetal tissues at 19 weeks of gestation using Integrate Data. Visual analysis revealed that the integrated cells could be divided into 11 cell populations. For each cluster, marker genes were explored and annotated to specific cell types. Based on published literature references, the cell populations were defined as mesenchymal cells, hematopoietic endothelial cells, immature thymic epithelial cells, pericytes, neural-associated cells, vascular smooth muscle cells, NKT cells, endothelial cells, fibroblasts, thymic epithelial cells-1, and thymic epithelial cells-2 (Figure 5C).

[0184] The results show that, compared with the natural thymus, the thymic organoids are highly similar to the cellular composition of the 19-week-old natural thymus fetus, with only a few cells showing numerical differences, such as fewer hematopoietic endothelial cells and a higher proportion of mesenchymal cells in the thymic organoids (Figure 5D).

[0185] Example 4: Co-culture of 3D thymus organoids derived from hPSCs with artificial hematopoietic stem / progenitor cells (HSPCs) to generate T cells in vitro.

[0186] S1.CD34 + Establishment of a co-culture system for artificial blood stem / progenitor cells (HSPCs) and thymus organoids

[0187] One day prior to the experiment, OP9 cells were seeded into 0.1% gelatin-coated six-well plates to achieve a density of 70-80%. OP9 cells were treated with mitomycin C before use; 3D thymus organoids (hETO) were washed three times with PBS and purified human umbilical cord blood CD34 by magnetic bead sorting. + Mix the HSPCs, centrifuge, discard the supernatant, and resuspend the pellet in 20 μL of culture medium. Use a 20 μL pipette tip to pick up the cell mixture and gently deposit it onto a 0.4 μm Millicell transwell insert (EMD Millipore) by forming a droplet at the tip. Place the insert on a six-well plate with OP9 cells to form two chambers, with 1 mL of mRB27 medium in the lower chamber of each well. Transfer the insert to a new monolayer of OP9 matrix cells every 4-5 days. Collect single cells from the upper chamber surrounding the thymus organoids every two weeks, filter hETO using a 40 μm cell filter, and harvest the single cell suspension for flow cytometry analysis. Culture for at least 6 weeks, changing the medium daily.

[0188] The mRB27 medium consists of RPMI 1640 (Thermo), 4% B-27 additive (Gibco), with the addition of 30 μM L-ascorbic acid-2-phosphate trisodium salt (Sigma), 1% penicillin-streptomycin-gentamicin mixture (Solarbio), 1% Glutamax (Thermo), 5 ng / mL FLT3L (Peprotech), 5 ng / mL IL-7 (Peprotech), and 10 ng / mL DLL4 (Peprotech). The medium needs to be freshly prepared weekly.

[0189] Thymus organoids and HSPCs were co-cultured in organoid culture chambers to establish a gas-liquid culture interface-induced T cell lineage typing method (Figure 6A). CD45 + At week two, the proportions of HSPCs cultured alone and HSPCs co-cultured with hETO were 97.1% and 96.0%, respectively. At week four, CD45... + The proportion of cells reached over 98% (Figure 6B). CD5 + CD7 + pro-T lineage cells appeared in the second week, and co-culture of HSPCs with hETO increased CD5 levels. + CD7 + The proportion of T cells reached its peak at 60.3% in week four, and then declined as T cells differentiated and matured (Figure 6C). More mature CD3 cells... + TCRαβ + T cells appear as early as week 2 and increase over time.

[0190] Compared to the HSPCs-only group, the hETO group induced more mature CD3. + TCRaβ + The proportion of T cells was high, reaching 11.5% at week six. The HSPCs group produced CD3+ at week six. + TCRaβ + The proportion of T cells was only 2.62% (Figure 6D).

[0191] CD3 at 2, 4 and 6 weeks of differentiation + TCRaβ + The proportion of T cells was statistically analyzed, and the results showed that at each time point, the hETO group had a lower CD3 count compared to the HSPCs group. + TCRaβ + The proportion of T cells was consistently high (Figure 6E). Immunofluorescence showed that HSPCs differentiated into CD3+ cells. + T cells (Figure 6F).

[0192] During the differentiation of HSPCs into the T lineage, the hETO group already showed 16.1% CD3 concentration by week 4. + TCRaβ + CD4 single positive (SP)T and 9.52% CD3 + TCRaβ + CD8 SP T cells were preferentially differentiated from HSPCs; at week 6 of differentiation, hematopoietic stem cells co-cultured with thymus organoids were more likely to be induced to differentiate into mature CD3 cells. + TCRαβ + CD4 SP T cells accounted for 90.3%, while only HSPCs showed a relatively high tendency to differentiate into mature CD3 cells in this differentiation system. + TCRαβ + CD8 SP T cells accounted for 8.5%, and in both the HSPCs group and the hETO group, more than 90% of CD3 cells were eliminated. + TCRαβ + T-cell positivity was selected as CD4 SP or CD8 SP T cells (Figure 6G).

[0193] In the thymus, CD3 + TCRαβ + CD8 SP and CD3 + TCRαβ + CD4 SP T cells from double-positive (DP)-like "immature" "Transformed into "mature" Phenotype. CD3 was detected at week 6. + TCRαβ + CD45RA + CD45RO - CCR7 + Mature The proportion of T, hETO group CD4 SP and The proportion of CD8 SPs in the hETO group was higher than that in the HSPCs group (P<0.05) (Figure 6H). The proportion of CD3 in the hETO group was also higher. + TCRγδ + The T cell ratio remained highly consistent throughout the experiment, reaching 10.9% at week 6, compared to only 1.76% in the HSPCs group, further demonstrating that thymic organoids can promote the multi-lineage differentiation potential of HSPCs into the T lineage (Figures 6I and 6J). At week 6 of differentiation, CD4+ in the hETO group... + CD25 + T in CD45 + CD3 + TCRαβ +The cell ratio was as high as 82.3%, while that of the HSPCs group was only 0.66%, which further proves that thymic organoids can promote the differentiation of HSPCs into the Treg lineage and have immune tolerance function (Figure 6K).

[0194] In summary, this in vitro T cell differentiation method provides a thymus-like developmental site for the formation of T lineages and can generate a very high proportion of mature CD3+ cells in vitro. + TCRαβ + CD4 SP T cells overcome the previous limitation of only being able to produce mature CD8 SP cells in vitro. + The bottleneck of T cells. This also aligns with the maturation of CD4 cells in the natural thymus and the internal environment. + T is higher than CD8 + T ratio relationship.

[0195] Example 5: Establishment of a method for thymus and immune system reconstruction in severely immunodeficient mice using hPSC-derived 3D thymus organoid transplantation.

[0196] NSI (Shenzhen In vivo Biomedical Technology Co., Ltd.), NCG, and NCG-X (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) in umbilical cord blood CD34 of severely immunodeficient mice + Sublethal irradiation (1.0-1.5 Gry) was administered to each mouse 6-8 hours before cell transplantation, and each mouse was transplanted with 300,000-500,000 CD34 cells. + Human umbilical cord blood hematopoietic stem / progenitor cells were used to perform thymic organoid renal capsule transplantation in mice one month later. Mice were anesthetized with isoflurane using an anesthesia machine. Hair was removed and the skin was prepared with povidone-iodine. The skin and muscles of the mice were incised to expose the kidneys, and multiple thymic organoids were implanted under the renal capsule. The kidneys were repositioned, and the muscles and skin were sutured and the wound disinfected. Mice were given 0.02% neomycin in their drinking water for one week. Aseptic techniques were maintained throughout the procedure, and the organs were kept moist.

[0197] Mice were euthanized after blood was collected from their tail veins, and their spleen, kidney grafts, and other tissues were harvested. After washing with PBS, the cells were homogenized using a 200 μm mesh, and the cell suspension was collected. Mice with CD45 were excluded through strict gating using a negative control. + Potential cell contamination. After washing the cell suspension, mouse erythrocytes were removed using erythrocyte lysis buffer, followed by flow cytometry staining for 30 minutes, and then washing once with PBS before analysis. Mice with transplanted human fetal thymus served as positive controls. Human CD3 levels in peripheral blood of mice were measured at 8 weeks (Fig. 7A), 12 weeks (Fig. 7B), and 16 weeks (Fig. 7C) after HSPC transplantation. + The proportion of T cells. Results showed that in mice transplanted with thymus organoids (hETO group), peripheral blood human CD3...+ The proportion of T cells was very close to that in mice that received transplants of natural human fetal thymus (Thymus group).

[0198] Cell flow cytometry analysis of hETO group severely immunodeficient mice 11 months after HSPC transplantation revealed that human CD45 was present in the bone marrow of the hETO group. + The cell proportion was 50.3%, CD3 + TCRαβ + The proportion of CD4SP T cells was 21.0%, CD4SP T cells were 18.7%, and CD8SP T cells were 15.0%. In addition, CD25... + FOXP3 + Treg cells account for 45% of CD45 + CD3 + CD4 + 10.2% of T cells, CD3 + TCRγδ + The proportion of T cells was 3.86% (Figure 7D). Human CD45 in the spleen... + The cell proportion was 75.9%, CD3 + TCRαβ + The proportion of CD4 SPT cells was 8.98%, CD4 SPT cells accounted for 6%, and CD8 SPT cells accounted for 58.2%. In addition, CD25... + FOXP3 + Treg cells account for 45% of CD45 + CD3 + CD4 + 7.28% of T cells, CD3 + TCRγδ + The proportion of T cells was 1.71% (Figure 7E).

[0199] Example 6: The human immune system reconstructed from severely immunodeficient mice derived from hPSC-derived 3D thymus organoid transplantation possesses humoral immunity capabilities.

[0200] To examine the functionality of reconstituted T cells in mice, human T lymphocytes from transplanted mice were examined, and their phenotypic and functional characteristics were compared with those of T cells generated after transplantation of natural human fetal thymus.

[0201] Mice were divided into four groups: blank control group (NC group), human umbilical cord blood HSPCs transplantation group (HSPCs group), human umbilical cord blood HSPCs transplantation and natural thymus group (Thymus group), and human umbilical cord blood HSPCs transplantation and thymus organoid transplantation group (hETO group). Each group of mice was then inoculated with chicken ovalbumin OVA antigen. The levels of ovalbumin-specific human immunoglobulins M and G (IgM and IgG) in the peripheral blood serum of mice were measured in the first week (Figure 8A) and the second week (Figure 8B) after OVA reimmunization.

[0202] The results showed that, compared with the Thymus group, higher levels of IgG and IgM were also detected in the peripheral blood serum of mice in the hETO group, indicating that thymic organoids and T cells domesticated from the natural thymus played a role in the synergistic process of B cell-mediated immunoglobulin class switching and antibody secretion, inducing a functional humoral immune response to protein antigens.

[0203] Example 7: The human immune system reconstructed from severely immunodeficient mice derived from hPSC-derived 3D thymus organoid transplantation possesses cellular immunity capabilities.

[0204] To verify the ability of T cells to reject allogeneic skin and form a memory response in vivo, allogeneic skin from C57BL / 6 mice was transplanted into mice reconstituted with human T cells. Flow cytometry analysis showed that a large number of mature human CD8SP T cells and CD4SP T cells were generated in the peripheral blood of the allogeneic skin transplant recipient mice (Figure 9A).

[0205] Six days after mouse skin transplantation, inflammatory cell infiltration in the transplanted skin grafts was detected using H&E and immunohistochemical staining. H&E staining showed that the dermis of the transplanted skin grafts from the hETO co-transplantation group (hETO group) and the natural thymus co-transplantation group (Thymus group) showed a large number of inflammatory cell infiltrations and larger necrotic areas. In contrast, the HSPCs transplantation group alone showed relatively fewer inflammatory cells (Figure 9B).

[0206] Similarly, immunofluorescence staining results also showed that CD3 in the hETO group skin grafts was present. + There was significant infiltration of T lymphocytes, and most of them expressed IL-10 (Figure 9C).

[0207] Peripheral blood samples were collected from mice to detect the proportions of human IL-2, IFN-γ, and TNF-α. The results showed that the hETO group mice produced a large number of cytokines, including IFN-γ. + TNF-α + The proportion was as high as 78.1%, IL2 + TNF-α + and IL-2 + IFN-γ +The proportions were similar to those in the Thymus group, at 86.6% and 76.2%, respectively. These results indicate that T cells acclimated through thymus organoids are capable of producing large amounts of cytokines to reject allogeneic skin grafts (Figure 9D).

[0208] In summary, these results demonstrate that reconstituted human T cells in mice mediated rejection of allogeneic skin grafts and stimulated T cell proliferation and activation, proving that a typical adaptive immune response can occur in mice with a humanized immune system.

[0209] Comparative Example: A Method for Directed Differentiation of Human Pluripotent Stem Cells (hPSCs) into Thymus Organoids via 3D Suspension Culture

[0210] Specifically, it includes the following steps (mesoendodermal cell differentiation stage):

[0211] The culture medium formulation is set as follows:

[0212] Method 1: Endoderm differentiation medium (X-VIVO 20 medium containing 100 ng / mL Activin A, 10 μM Y-27632).

[0213] Method 2: Endoderm differentiation medium-1 (X-VIVO 20 medium containing 100 ng / mL Activin A, 50 ng / mL Wnt3A, 10 μM Y-27632) and endoderm differentiation medium-2 (X-VIVO 20 medium containing 1×B-27 additive, 100 ng / mL Activin A).

[0214] Method 3: Endoderm differentiation medium-1 (X-VIVO 20 medium containing 100 ng / mL Activin A, 5 μM CHIR99021, 10 μM Y-27632) and endoderm differentiation medium-2 (X-VIVO 20 medium containing 1×B-27 additive, 100 ng / mL Activin A).

[0215] S1. Cell clump digestion of hPSCs: After aspirating the culture medium, wash the cells with 1 mL of PBS, aspirate the PBS, add 1 mL of GCDR digestion solution, and incubate at 37°C in a CO2 incubator for 3 minutes. Then, aspirate the GCDR from the wells, add day 0 endoderm culture medium, and gently pipette the cells until small cell clumps are formed, finally obtaining a cell suspension. Seed the cell suspension in low-adhesion six-well plates, add culture medium, mix well, and incubate.

[0216] S2. Cell medium change: Collect cell spheres into centrifuge tubes, use natural sedimentation or low-speed centrifugation to sink the cell spheres to the bottom of the tube, discard the supernatant, add 1 mL PBS to wash the human pluripotent stem cell spheres, discard the supernatant, and then add the endoderm differentiation medium at the corresponding time point.

[0217] Method 1 uses 100 ng / mL Activin A and 10 μM Y-27632 from Day 0 to Day 3 (labeled as Method A). Method 2 uses 100 ng / mL Activin A, 50 ng / mL Wnt3A and 10 μM Y-27632 from Day 0 to Day 1, and uses 1×B-27 culture medium supplement and 100 ng / mL Activin A from Day 2 to Day 3 (labeled as Method A+B+W). Method 3 replaces Wnt3A in Method 2 with 5 μM CHIR99021 (labeled as Method A+B+C).

[0218] S4. Cell Harvesting: On day 3 of culture, endoderm cell spheres were analyzed by flow cytometry, immunofluorescence, and RT-qPCR. Endoderm cell spheres were collected, allowed to settle naturally, and the supernatant was discarded. The spheres were then washed with 1 mL of PBS. RNA extraction and RT-qPCR were subsequently performed.

[0219] During this differentiation process, we strive to find an optimal endoderm-promoted thymus organoid differentiation pathway by exploring three endoderm differentiation methods.

[0220] Cell spheres were collected from Day 0 to Day 3. qRT-PCR was used to detect the mesodermal marker gene Brachyury and the endoderm marker genes SOX17, FoxA2, ​​and CXCR4. Results showed no significant difference in Brachyury mRNA expression levels at Day 3. In differentiation systems with Wnt3A or the small chemical molecule CHIR99021, the relative expression levels of SOX17, FoxA2, ​​and CXCR4 were slightly higher than in the first combination (Method A) (Figure 10). As differentiation progressed, organoids from Day 17 were collected to detect specific markers of epithelial cells (EpCAM, K8, K5, AIRE), mesenchymal cells (PDGFRA), endothelial cells (PECAM1), and smooth muscle cells (ACTA2) in the human thymus. Results showed that the third endoderm differentiation method (Method A+B+C) had a greater potential advantage for the development and maturation of thymus organoids (Figure 11).

[0221] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A culture medium combination comprising a first culture medium and a second culture medium; the first culture medium comprising a first basal culture medium supplemented with Activin A, a GSK-3 inhibitor and a ROCK inhibitor; and the second culture medium comprising a second basal culture medium supplemented with Activin A and B-27 additives.

2. The culture medium combination according to claim 1, characterized in that, The GSK-3 inhibitor is selected from at least one of B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHIR-99021; preferably, the GSK-3 inhibitor is CHIR99021. Preferably, the ROCK inhibitor is selected from at least one of Y-27632, Thiazovivin, FasudilHCl, GSK429286A, RKI-1447 and Azaindole 1; preferably, the ROCK inhibitor is Y-27632. Preferably, the concentration of Activin A added to the first basal culture medium is 80-120 ng / mL; Preferably, the concentration of CHIR99021 added to the first basal culture medium is 4-6 μM; Preferably, the concentration of Y-27632 added to the first basal culture medium is 8-12 μM.

3. The culture medium combination according to claim 1, characterized in that, The concentration of Activin A added to the second basal culture medium is 80-120 ng / mL; Preferably, the concentration of B-27 additive in the second basal culture medium is 0.5-1.5×.

4. The culture medium combination according to claim 1, characterized in that, The culture medium combination also includes a third culture medium; the third culture medium includes a third basal culture medium supplemented with a TGF-β signaling pathway inhibitor and a Notch receptor agonist; Preferably, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; preferably, the TGF-β signaling pathway inhibitor is SB431542; Preferably, the Notch receptor agonist is selected from DLL4; Preferably, the concentration of SB431542 added to the third basal culture medium is 8-12 μM; Preferably, the concentration of DLL4 added in the third basal culture medium is 8-12 ng / mL.

5. The culture medium combination according to claim 1, characterized in that, The culture medium combination also includes a fourth culture medium, which includes a fourth basal culture medium supplemented with TGF-β signaling pathway inhibitors, Notch receptor agonists, and cytokines; Preferably, the TGF-β signaling pathway inhibitor is selected from at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; preferably, the TGF-β signaling pathway inhibitor is SB431542; Preferably, the Notch receptor agonist includes DLL4; Preferably, the cytokines include FLT3L, TPO, and SCF; Preferably, the concentration of SB431542 added to the fourth basal culture medium is 8-12 μM; Preferably, the concentration of DLL4 added in the fourth basal culture medium is 8-12 ng / mL; Preferably, the concentration of TPO added to the fourth basal culture medium is 4-6 ng / mL; Preferably, the concentration of FLT3L added to the fourth basal culture medium is 4-6 ng / mL; Preferably, the concentration of SCF added in the fourth basal culture medium is 40-60 ng / mL.

6. The culture medium combination according to any one of claims 1-5, characterized in that, The first and second basal culture media are stem cell culture media; The third and fourth basic culture media are endothelial cell culture media, and the endothelial cell culture media are selected from any one of EGM2 medium, ECM medium, and VascuLife VEGF Medium Complete Kit medium; Preferably, the first basal culture medium and the second basal culture medium are X-VIVO 20 culture medium; Preferably, the third and fourth basic culture media are EGM2 culture media.

7. A reagent kit, characterized in that, The kit comprises the culture medium combination according to any one of claims 1-6; Preferably, it also includes trophoblast cells.

8. The application of the culture medium combination according to any one of claims 1-6 or the kit according to claim 7: (1) Preparation of mesoderm cells; (2) Preparation of endoderm cells from the third pharyngeal pouch; (3) Preparation of thymus organoids; (4) Prepare products that induce pluripotent stem cells to differentiate into mesoendothelial cells; (5) Prepare products that induce pluripotent stem cells to differentiate into third pharyngeal pouch endoderm cells; (6) Prepare products that induce pluripotent stem cells to differentiate into thymic organoids.

9. A method for preparing mesoderm cells and / or third pharyngeal pouch endoderm cells and / or thymus organoids, wherein mesoderm cells and / or third pharyngeal pouch endoderm cells and / or thymus organoids are obtained by culturing pluripotent stem cells using the kit described in claim 7; wherein the culture is a suspension culture; Preferably, the method specifically includes: (1) Pluripotent stem cells were cultured successively in the first culture medium and the second culture medium in the kit described in claim 7 to obtain mesoendodermal cells; and / or (2) Third pharyngeal pouch endoderm cells are obtained by co-culturing mesoendodermal cells and trophoblast cells in the third culture medium of the kit described in claim 7; and / or (3) Thymoid organoids were obtained by culturing the third pharyngeal endoderm cells in the fourth culture medium of the kit of claim 7.

10. The use of the thymus organoid prepared by the method of claim 9 in any of the following: 1) Used to construct humanized blood and / or immune system animals; 2) Products used to prepare animals with humanized blood and / or immune systems; 3) Simulate human diseases; 4) Used to prepare products that mimic human diseases; 5) Preparation of human T cells; 6) Products used to prepare human T cells; 7) Used to prepare products that induce transplant tolerance; 8) Used to prepare products that restore thymus function; 9) Generates humanized antibodies; 10) Used to generate humanized antibodies; 11) Thymus-related drug screening, development, and / or toxicity analysis; 12) Investigate the pathogenesis of thymic diseases; 13) Prepare products for treating thymic diseases; 14) Construct a thymus disease model; 15) Thymus toxicology test.

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