Induced pluripotent stem cell capable of overcoming immunogenicity as well as establishment method and application thereof

Abstract

The invention discloses an establishment method of induced pluripotent stem cells capable of overcoming immunogenicity and derived mesenchymal stem cells of the induced pluripotent stem cells, belongs to the technical field of biology, and solves the problems of limited passage times, large individual difference and immunological rejection of the mesenchymal stem cells in the prior art. The method comprises the following steps: reprogramming starting somatic cells to establish an induced pluripotent stem cell line, and continuously picking monoclonal passage to obtain a monoclonal induced pluripotent stem cell line; and performing beta2-microglobulin knockout on the monoclonal induced pluripotent stem cell strain to obtain beta2-microglobulin knockout induced pluripotent stem cell monoclone, namely the induced pluripotent stem cell capable of overcoming immunogenicity. The invention develops a method for inducing wild type and B2M knockout type hiPSC to differentiate into the iMSC by regulating an embryonic development signal channel to generate the iMSC, and the differentiated iMSC-WT and iMSC-KO are extremely similar to UCMSC in phenotype.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to an induced pluripotent stem cell that overcomes immunogenicity, its establishment method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with significant research and application value in regenerative medicine and immunomodulation. Cell-mediated allogeneic immune rejection limits the persistence of MSCs in vivo, thus limiting their therapeutic potential. Therefore, further reducing the immunogenicity of MSCs to improve survival time after allogeneic MSC transplantation, thereby enhancing therapeutic efficacy and achieving long-term clinical benefits, has become a highly significant research topic. Studies show that human leukocyte antigen (HLA) class I and II loci play important roles in adaptive and innate immunity; HLA-I, composed of heavy and light chains, is also known as β2-microglobulin (B2M). When the B2M gene is deleted, the HLA-I structure is disrupted and it becomes non-functional.

[0003] However, it is almost impossible to reliably obtain B2M knockout adult MSCs because adult-derived MSCs have limited proliferation capacity. Knocking out the B2M gene for each adult MSC would be extremely costly, and it would be difficult to guarantee quality consistency, and it would also increase the complexity of CMCs. Summary of the Invention

[0004] This invention provides an induced pluripotent stem cell that overcomes immunogenicity, along with its establishment method and application, to address the problems of limited passage times and significant individual variability in existing mesenchymal stem cells.

[0005] In a first aspect, the present invention provides a method for establishing induced pluripotent stem cells that overcome immunogenicity, comprising the following steps: establishing an induced pluripotent stem cell line by reprogramming starting somatic cells; obtaining a monoclonal induced pluripotent stem cell line by continuously selecting and passaged monoclonal cells, wherein the monoclonal induced pluripotent stem cell line is genotype-matched to the parental cells; knocking out β2-microglobulin in the monoclonal induced pluripotent stem cell line to obtain a β2-microglobulin knockout induced pluripotent stem cell monoclonal, i.e., an induced pluripotent stem cell that overcomes immunogenicity.

[0006] As one possible implementation, the starting somatic cells are umbilical cord blood mononuclear cells; and / or, the reprogramming is performed using the CTS Cytotune Sendai reprogramming kit; and / or, the β2-microglobulin knockout is performed using CRISPR-Cas9 technology.

[0007] Umbilical cord blood cells (UCB) are an ideal donor cell type for generating hiPSCs. Umbilical cord cells are the youngest cells that can be collected non-invasively and can be stored for decades without losing viability. The Sendai virus reprogramming system requires no special equipment or reagents and offers advantages such as non-integration, easy removal, high reprogramming efficiency, and low aneuploidy rate, significantly reducing CMC complexity. Therefore, in this invention, considering factors such as reprogramming efficiency, aneuploidy rate, and potential pharmaceutical translation applications, a CTS-level Sendai virus reprogramming system is used to obtain hiPSCs. This invention uses Thermo's commercially available B2M TrueGuide to synthesize gRNA and RNPs formed from TrueCut Cas9 protein, which are then introduced into hiPSCs via electroporation. This process eliminates transcription and translation steps, significantly improving Cas9-mediated editing efficiency. Furthermore, compared to transfecting Cas9 plasmids (which typically remain in cells for more than 72 hours and may exacerbate off-target cleavage effects), TrueCut Cas9 protein is cleared from cells within 24 hours after electroporation, which greatly reduces off-target effects.

[0008] In a second aspect, the present invention provides an induced pluripotent stem cell that overcomes immunogenicity obtained by the establishment method described in the first aspect.

[0009] As one possible implementation, it exhibits normal pluripotency and normal differentiation ability in its 15th generation and earlier.

[0010] In this invention, normal pluripotency refers to a positive cell proportion of more than 90% expressing pluripotency markers NANOG, SOX2, OCT4, SSEA4, TRA-1-60, and TRA-1-81, and normal differentiation capacity refers to the ability to differentiate into the three germ layers: endoderm, mesoderm, and ectoderm. Correspondingly, at least two of the above-mentioned pluripotency markers are not expressed satisfactorily, or a certain germ layer cannot differentiate.

[0011] Thirdly, the present invention provides a mesenchymal stem cell obtained by inducing differentiation of immunogenic induced pluripotent stem cells as described in any possible implementation of the second aspect.

[0012] Despite extensive research and promising results in regenerative medicine, disease modeling, and therapy, mesenchymal stem cells (MSCs) face significant obstacles to mainstream clinical application. These obstacles include the unsustainable expansion of adult MSCs in vitro and the considerable heterogeneity among MSCs from different donors and tissues, making it difficult to obtain sufficient numbers of therapeutic cells from a single donor. It is well known that MSC expansion in culture accelerates their senescence, leading to decreased proliferative capacity and phenotypic changes. Therefore, obtaining an unlimited number of homogeneous MSCs in vitro is of great value to both basic and applied sciences. Unlike tissue-derived MSCs, iMSCs represent a new, reliable, and unrestricted source. iMSCs can be generated from homogeneous and well-characterized hiPSC cell lines, allowing for faster proliferation, slower senescence, and easier genome editing, thus mitigating many of the aforementioned technical and biological limitations. Furthermore, the use of hiPSCs avoids the ethical issues surrounding the use of human embryonic stem cells.

[0013] As one possible implementation, the induction differentiation step includes: resuscitating and passaged the immunogenic induced pluripotent stem cells and then placing them in a directed differentiation medium for directed differentiation culture, and passaged them to the first generation of differentiated cells; passaged the first generation of differentiated cells to the fifth generation to obtain the mesenchymal stem cells.

[0014] This invention characterized the obtained iMSC-WT and iMSC-KO according to the MSC criteria proposed by the International Society for Cell and Gene Therapy (ISCT). The results showed that iMSC-WT and iMSC-KO were very similar to UCMSCs in terms of cell morphology, surface marker expression, and trilineage differentiation capacity. Furthermore, this invention evaluated the cytokine secretion capacity and inhibitory capacity of iMSCs against Bv2 cell proliferation, showing no significant difference compared to UCMSCs. Further, this invention used single-cell sequencing technology to compare the differences between iMSCs and UCMSCs at a more refined level. The results showed no overlap in single-cell subsets between iMSCs and UCMSCs, but the cell type of iMSCs was unsupervisedly annotated as "MSC," while UCMSCs carried some gene expression characteristics of umbilical vein smooth muscle cells, reflecting the differences between the two. Pseudo-temporal analysis showed that iMSCs are in an earlier stage of development, with higher activity in signaling pathways such as cell cycle, mesenchymal stem cell development, and differentiation than UCMSCs, but slightly lower activity in signaling pathways such as extracellular matrix and migration. These results indicate that the iMSCs obtained by the differentiation scheme of this invention conform to the identity of MSCs and are more primitive and have higher differentiation potential than UCMSCs.

[0015] Fourthly, the present invention provides a directional differentiation culture medium as described in the third aspect, comprising the following components: First part: StemFit basic 03-A basal medium and StemFit basic 03-B thawed overnight at 2–8°C, mixed thoroughly in a biosafety cabinet, and dispensed into 50 mL centrifuge tubes; CHIR99021 to 3 μM and SB431542 to 10 μM are added; Second part: TBD culture medium additive thawed overnight at 2–8°C, TBD basal medium and TBD additive mixed thoroughly in a biosafety cabinet, and dispensed into 50 mL centrifuge tubes; CHIR99021 to 3 μM and SB431542 to 10 μM are added.

[0016] This invention achieves the directed induction of hiPSCs into iMSCs based on the directed differentiation culture medium composed of the first and second parts. It adopts an embryoid approach and combines small molecule compounds to regulate germ layer development-related signaling pathways. Specifically, it activates the WNT signaling pathway based on the GSK3β inhibitor CHIR99021 and simultaneously uses the TGFβ signaling pathway inhibitor SB431542 to directed induction of hiPSCs into mesenchymal cell-like iMSCs.

[0017] Furthermore, the first part, after adding SB431542 to 10 μM, also includes adding BMP4 to 50 ng / mL; the second part, after adding SB431542 to 10 μM, also includes adding L-Ascorbic acid to 100 μM.

[0018] The inventors further discovered in their research that by introducing BMP4 into the first part of the culture medium and L-Ascorbic acid into the second part of the culture medium, they can fully utilize the properties of BMP4 to promote the differentiation of iPSCs into mesoderm and the properties of L-Ascorbic acid to promote the differentiation of iPSCs into mesoderm and the proliferation of iMSCs, thereby achieving the directed induction of hiPSCs into mesenchymal stem cells (iMSCs).

[0019] Fifthly, the present invention provides the use of immunogenic induced pluripotent stem cells as described in any possible implementation of the third aspect or mesenchymal stem cells as described in any possible implementation of the fourth aspect in the preparation of medicaments for transplantation therapy.

[0020] This invention develops a method to generate iMSCs by inducing the differentiation of wild-type and B2M knockout hiPSCs into iMSCs through regulation of embryonic developmental signaling pathways. The differentiated iMSC-WT and iMSC-KO cells are phenotypically very similar to UCMSCs. Single-cell transcriptome analysis revealed that the iMSCs are in a more primitive MSC state, with superior proliferation and differentiation potential compared to UCMSCs. This invention also demonstrates the low immunogenicity of iMSC-KO, providing an ideal cell source for subsequent stem cell transplantation therapy research.

[0021] Induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) offer numerous advantages and are increasingly being used in basic and clinical research. First, iMSCs are more primitive in development, possessing stronger self-renewal, proliferation, and differentiation potential. Reports indicate that iMSCs can differentiate into bone, cartilage, and adipocytes in vitro, and also acquire a secretome similar to their parental adult mesenchymal stem cells (MSCs), with enhanced paracrine capabilities. Second, compared to hepatoid cells (hiPSC-HLCs) differentiated from hiPSCs, iMSCs have a shorter differentiation time, are simpler to differentiate, are easier to mass-produce, and have a smaller diameter than adult MSCs, thus reducing the risk of embolism during intravenous infusion. Finally, compared to adult MSCs, iMSCs are not limited by donor age. These cells express a large number of genes that play important roles in embryonic tissue and development, which endow iMSCs with high proliferative capacity and regenerative properties. iMSCs can maintain their proliferation and differentiation potential even after long-term in vitro culture (more than 10 passages) without affecting their therapeutic efficacy. In contrast, adult MSCs enter a senescent stage with increasing passage numbers, exhibiting genomic instability, accumulation of DNA damage, altered DNA repair pathways, increased oxidative stress damage, metabolic homeostasis dysregulation, and activation of immune rejection, with significantly weakened differentiation potential and function. Therefore, iMSCs may have broader application potential.

[0022] The B2M knockout hiPSCs (hiPSC-KO) established in this invention do not express HLA-I class I molecules but maintain their pluripotency and genomic stability. Furthermore, iMSCs (iMSC-KO) are differentiated from hiPSC-KO in this invention. It has been further demonstrated that B2M knockout does not affect the iMSC phenotype, differentiation potential, or immunosuppressive properties, and it has also been confirmed that no B2M protein is expressed in the B2M knockout hiPSC-derived iMSCs. In vitro cytotoxicity assays showed a large release of IFNγ from allogeneic reactive NK cells after stimulation with iMSC-WT, while iMSC-KO significantly reduced this phenomenon. This in vitro study reveals the privilege of iMSC-KO in allogeneic NK cell-mediated immune rejection responses. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A represents the hiPSC cloning process obtained from UCBMC through reprogramming according to an embodiment of the present invention, wherein A represents representative hiPSC images (Scale bar = 200 μm) at the 3rd and 15th generations on the 20th day after reprogramming. Figure 1 B represents the flow cytometry results of the clinical-grade hiPSC pluripotency markers NANOG, OCT4, SOX2, TRA-1-60, TRA-1-81, and SSEA4 provided in the embodiments of this invention. Figure 1 C represents the clinical-grade hiPSC trigerm layer differentiation ability detection results provided in this embodiment of the invention, wherein the immunofluorescence staining of three germ layer markers is performed: endoderm marker SOX17, mesoderm marker BRACHYURY, and ectoderm marker β3-TUBLIN (Scale Bar = 200 μm).

[0025] Figure 2 The following diagrams illustrate the B2M knockout efficiency and single-clone knockout verification provided in this embodiment of the invention. A represents the sgRNA cleavage efficiency results provided in this embodiment, where black arrows indicate the sgRNA direction and red arrows indicate the cleavage sites. B shows the first-generation sequencing map of single clone #3; C shows the first-generation sequencing map of single clone #18; and D shows the first-generation sequencing map of single clone #22.

[0026] Figure 3For the hiPSC-KO quality detection – pluripotency, karyotype, and differentiation capacity provided in this embodiment of the invention, A is the morphology of hiPSC-Bko-3; B is alkaline phosphatase staining; C is chromosome karyotype; D is immunofluorescence staining for pluripotency markers, including double staining with NANOG and TRA-1-81, and double staining with SOX2 and OCT4; Scale Bar = 200 μm; E is the detection of trigerm layer differentiation capacity, Scale Bar = 200 μm.

[0027] Figure 4 The phenotypic identification results of UCMSCs provided in the embodiments of the present invention are shown in Figure A, which is the flow cytometry detection result of UCMSC surface markers; and Figure B is the three-lineage differentiation staining result. Left: Alizarin staining image after osteogenic differentiation (Scale bar=50 μm), middle: Alicin blue staining image after chondrogenic differentiation (Scale bar=200 μm); right: Oil Red O staining image after adipogenic differentiation (Scale bar=100 μm).

[0028] Figure 5 The iMSC differentiation protocol and cell morphology at different differentiation stages provided in this embodiment of the invention are shown in Figure A. A represents the iMSC differentiation protocol. B represents the cell morphology observations on day 10 of iMSC differentiation, day 6 of P0, and day 3 of P3 generation. C represents the morphology of iMSC-WT and iMSC-KO before passage at P3 and P5 (Scale bar = 200 μm).

[0029] Figure 6 The present invention provides flow cytometry analysis of surface marker expression of iMSC-WT and iMSC-KO; wherein, A is the flow cytometry analysis result of iMSC-WT; and B is the flow cytometry analysis result of iMSC-KO.

[0030] Figure 7 A shows the comparison results of the three-lineage differentiation of iMSC-WT and iMSC-KO provided in the embodiments of the present invention. Among them, left: Oil Red O staining image after adipogenic differentiation (Scale bar = 100 μm); middle: Alizarin staining image after osteogenic differentiation (Scale bar = 50 μm); right: Alicin Blue staining image after chondrogenic differentiation (Scale bar = 200 μm). Figure 7 B and Figure 7 C represents the results of cytokine secretion detection and BV2 proliferation inhibition assay provided in the embodiments of the present invention, wherein B represents the content of four cytokines in the cell culture supernatant detected by ELISA (n≥3); and C represents the analysis of the inhibitory ability of three types of stem cells on BV2 cell proliferation (n = 9).

[0031] Figure 8A represents the karyotype analysis results provided in the embodiments of the present invention. Figure 8 B represents the soft agar colony formation assay, with a scale bar of 200 μm.

[0032] Figure 9 A and Figure 9 B represents the first-generation sequencing verification of the B2M gene knockout in iMSC-KO provided in this embodiment of the invention, wherein A is the first-generation sequencing diagram of the B2M gene locus in iMSC-WT; and B is the first-generation sequencing diagram of the B2M gene locus in iMSC-KO. Figure 9 C represents the iMSC-KO provided in this embodiment of the invention, which does not express the B2M protein (Scale bar = 100 μm).

[0033] Figure 10 The ELISA analysis provided in this embodiment of the invention provides the differences in the levels of IFNγ released from NK cells stimulated by three different cell types. . Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0036] Example 1

[0037] This embodiment provides a method for establishing induced pluripotent stem cells (hiPSCs) induced differentiation into mesenchymal stem cells (iMSCs), including:

[0038] 1. Isolation and culture of umbilical cord blood mononuclear cells:

[0039] UCBMC medium (prepared fresh, made by mixing StemPro™-34 medium and StemPro™-34 Nutrient supplement, then adding L-Glutamine 2 mM (final concentration), SCF 100 ng / mL (final concentration), IL-3 20 ng / mL (final concentration), IL-6 20 ng / mL (final concentration), and FLT-3Ligand 100 ng / mL (final concentration)) was equilibrated to room temperature. 9 mL of the medium was transferred to a 15 mL centrifuge tube and heated to 37 °C in a water bath. Umbilical cord blood cells were removed from liquid nitrogen and rapidly thawed in a water bath. The thawed cells were then slowly added along the wall of the centrifuge tube to a 15 mL centrifuge tube containing the aforementioned UCBMC medium. The mixture was gently shaken and centrifuged at 1500 rpm for 5 min at room temperature. The supernatant was discarded, and UCBMC medium was added and mixed to resuspend the cells, yielding a cell suspension. The cell suspension was transferred to a T25 cell culture flask, and the medium was brought to a final volume of 5 mL. The cells were cultured at 37°C and 5% CO2 for 3 days. During induction, the cells were observed and fresh UCBMC medium was added. Specifically, 2 mL of medium was gently aspirated from each bottle and 2 mL of fresh medium was added. If cells were present during aspiration, the cells were centrifuged at 1500 rpm for 5 min at room temperature, the supernatant was discarded, the cells were resuspended in 2 mL of fresh medium, and then the cells were added back to the culture system.

[0040] 2. Sendai virus infection:

[0041] Carefully aspirate the upper layer of culture medium containing suspended cells from the T25 culture flask, retaining the adherent cells at the bottom. Wash the bottom of the flask once with calcium- and magnesium-free DPBS, add 2 mL of TryPLE Express Enzyme to digest the cells for 5 min, collect the digested cells, centrifuge at 1500 rpm for 3 min at room temperature, resuspend the cells, and count them using the CountStar system. Take 1×10⁶ cells. 6Cell suspension was placed in 15 mL centrifuge tubes, centrifuged, and the supernatant was discarded. This was labeled as the centrifugation system. Three vials of CTS CytoTune 2.1 Sendai virus were added to 1 mL of UCBMC medium equilibrated to room temperature. hKOS, hL-Myc, and hKlf4 were added at an MOI of 5. The mixture was gently pipetted up and down to ensure complete mixing, yielding the Sendai virus solution. This Sendai virus solution was added to the centrifugation system and mixed with the cell suspension at the bottom of the tube. The mixture was centrifuged at 1000 × g for 30 min. The cells were resuspended in the supernatant and seeded into 6-well plates coated with Ln521. The plates were incubated at 37°C with 5% CO2. The supernatant was gently aspirated, and 1 mL of UCBMC medium was added to each well. If cells were present in the aspirated medium, the plates were centrifuged at 1500 rpm for 5 minutes at room temperature. After 5 min, discard the supernatant, resuspend the cells in fresh culture medium, and add the cells back to the culture plate. Culture at 37℃ and 5% CO2, changing the UCBMC medium every other day. On day 8 of infection, discard the medium from the wells, wash once with calcium- and magnesium-free DPBS, add 0.5 mL TryPLE Express Enzyme to digest the cells for 5 min, centrifuge at 1500 rpm for 5 min at room temperature, resuspend the cells, and count them. Discard the Ln521 coating medium from the 6-well plate, add 2 mL of E8 complete culture medium, and add the cells at 5 × 10⁶ cells / well. 4 / wells were inoculated into Ln521-coated 6-well plates and cultured at 37°C with 5% CO2. The E8 medium was then changed daily thereafter.

[0042] 3. Single-clone selection and subculturing:

[0043] Equilibrate the Ln521-coated 48-well plate and E8 complete medium to room temperature. Discard the coating medium from the Ln521-coated 48-well plate, replace 0.2 mL of E8 complete medium in each well, open the plate cap, and under a microscope, use a 1 mL syringe needle to peel morphologically sound clones from the plate surface. Then, use a 10 μL pipette to transfer the clones to 1.5 mL EP tubes containing 0.2 mL of E8 complete medium, pipette three times, and seed into the coated 48-well plate. Incubate in an incubator, changing the medium daily for 5-7 days until the hiPSC colonies are too dense or too large, or when the colony differentiation degree increases. Then, perform cell passage to obtain the monoclonal induced pluripotent stem cell line iPSC-WT. Cell passage includes: equilibrating the Ln521-coated cell culture plate and the required volume of E8 complete medium to room temperature, discarding the coating medium from the cell culture plate, and replacing 1 mL of E8 complete medium in each well. Discard the cell culture medium in the 48-well plate with E8 complete medium, wash once with DPBS without calcium and magnesium, add 0.2 mL ReleSR, incubate at room temperature for 3 min, add 0.3 mL E8 complete medium, gently pipette no more than 10 times, add cell cluster suspension at a volume ratio of 1:15, gently shake well and place in an incubator for culture, changing the medium daily with fresh medium.

[0044] 4. B2M gene knockout was used to establish a single-clone iPSC-KO induced pluripotent stem cell line with β2-microglobulin knockout:

[0045] sgRNA powder (purchased from Thermo Fisher, sgRNA targeting exon 2 of the B2M gene, sequence CGUCAUCCAGCAGAGAA) and 15 μL of RNase-free water were centrifuged at 4000 ×g for 5 min. The mixture was gently tapped against the tube wall to mix, and then rapidly centrifuged to allow the liquid to pool at the bottom of the tube. The mixture was incubated at room temperature for 30 min to obtain a 100 μM (100 pmol / μL) stock solution. This solution was aliquoted into 2 μL vials and stored at -20℃ (using the LONZA electroporation kit, 100 pmol per reaction, i.e., 1 μL). P15 generation iPSC-WT cells were digested into single cells using the dissociative enzyme Accutase and counted. Cells were then analyzed at 8 × 10⁻⁶ cells per cell. 5Centrifuge the cells, resuspend them in PBS, and wash three times to remove as much supernatant as possible, retaining the cell pellet. Resuspend the cell pellet in electroporation working solution (Nucleofector Solution, 82 μL; Supplement, 18 μL; sgRNA 100 pmol, 1 μL; Cas9 protein, 3 μL). Slowly add the cell suspension along the side wall of the electroporation cuvette (avoid creating air bubbles), cover, and transfer the cuvette to the electroporator (note the cuvette orientation). Start the program: LONZA→X→H9→start. Two samples can be electroporated simultaneously. After electroporation, remove the cuvette and aspirate the cells using a small Pasteur pipette. Seed the cells into two wells of a 6-well LN521-coated plate in SF03 + 10 μM medium. Y27632 culture medium was incubated at 37℃. The next day, the medium was replaced with SF03 medium without Y27632. When the cells reached 80%–90% confluence, they were digested into single cells and counted. At a rate of 3000 cells / well, two wells of a 6-well plate were seeded for picking single colonies. These were then cultured in SF03 + 10 μM Y27632 medium. A portion of the remaining cells were cultured at 2 × 10⁻⁶ cells / well. 5 Five vials were cryopreserved, and the remaining cells were divided into two portions. After centrifugation, one portion was stored at -80°C, and the other portion was sent for first-generation sequencing to verify the knockout efficiency. After 48 hours, the medium was replaced with Y27632-free medium for further culture. Cells were cultured for 4-5 days to observe the growth of small clones that did not come into contact with each other. Under a microscope, a single clone with good morphology was picked with a 10 μL pipette tip, pipetted 3 times, and then seeded into one well of a 24-well plate and cultured in SF03 + 10 μM Y27632. The next day, the medium was replaced with Y27632-free SF03. After 5-6 days of culture, once the clones had grown, they were digested into single cells and divided in half. One half was centrifuged and cryopreserved, and the other half was centrifuged and the pellet was sent for first-generation sequencing. All single clones that were successfully knocked out were revived into one well of a 6-well plate and cultured in SF03 + 10 μM Y27632. The SF03 medium was changed daily thereafter. When the cells reached 80% confluence, they were passaged at a ratio of 1:15-20 for subsequent library construction and quality testing.

[0046] 5. hiPSC culture and induced differentiation

[0047] iMSC-induction medium 1 (MIM-1): Thaw StemFit basic 03-A and StemFit basic 03-B overnight at 2–8°C, mix thoroughly in a biosafety cabinet, and aliquot into 50 mL centrifuge tubes; add CHIR99021 to 3 μM, SB431542 to 10 μM, and BMP4 to 50 ng / mL; the BMP4 product number is 314-BP-020 / CF, R&Dsystems.

[0048] iMSC-induction medium 2 (MIM-2): Thaw the TBD medium supplement overnight at 2–8°C. Mix the TBD basal medium and TBD supplement thoroughly in a biosafety cabinet and aliquot into 50 mL centrifuge tubes. Add CHIR99021 to 3 μM, SB431542 to 10 μM, and L-Ascorbic acid to 100 μM; the L-Ascorbic acid is HY-B0166, medchemexpress.

[0049] Revive the 18th generation hiPSC, with the generation number after revival being P19. Propagate twice consecutively until P21;

[0050] 1) Directed differentiation of hiPSC-MSC:

[0051] D-1: 21 generation hiPSC cells were grown in one well until they reached 80% confluence. The cells were then digested using ReleSR, and the cell clumps were resuspended in StemFitbasic 03 complete medium, supplemented with Y27632 to 10 μM. The cell clump suspension was transferred to a low-attachment 6 cm bacterial culture dish and cultured in suspension.

[0052] D0: After 24 hours, observe the formation of embryoid bodies (EB) under a microscope. Gently collect the EB suspension into a 15 mL centrifuge tube (Note: If there are adherent clumps or clusters, gently blow them to obtain smaller EBs). Let it stand at room temperature for 5 min to allow the EBs to settle naturally. Discard the supernatant, add 5 mL of DPBS to wash the EBs, gently resuspend them, and let them stand at room temperature for 3-5 min again to allow the EBs to settle naturally. Discard the DPBS, add MIM-1, and transfer the EB suspension to a new low-adhesion 6 cm bacterial culture plate for suspension culture.

[0053] D1, D2: Observe and change MIM-1 daily. When changing the solution, you can directly aspirate the supernatant, but be careful not to aspirate the EB.

[0054] D3: Observe EB formation under a microscope, collect cell cluster suspension into 15 mL centrifuge tubes (Note: If there are adherent clumps or aggregated clumps, gently pipette to obtain smaller EBs), let stand at room temperature for 3 min to allow EBs to settle naturally. Discard the supernatant, add 5 mL of DPBS to wash the EBs, aspirate the liquid to gently resuspend the EBs, let stand at room temperature for 3 min again to allow EBs to settle naturally, discard the DPBS, add MIM-2, transfer the EB suspension to a new low-adhesion bacterial culture plate, and culture in suspension.

[0055] D4~D5: Change MIM-2 daily. When changing the solution, you can directly aspirate the supernatant, but be careful not to aspirate the EB.

[0056] D6: Take one 6-well plate that has been pre-coated with LN521, warm it to room temperature for 30 min, discard the coating solution, add 2 mL of MIM-2 to each well for later use, gently pick up EB with a pipette, and seed the EBs into the LN521-coated culture plate at a rate of 50-100 EBs / well, gently shake the culture plate to distribute the EBs evenly, and incubate at 37℃.

[0057] D8: After 48 hours of culture, replace with fresh MIM-2 medium, being careful not to blow the EBs away when adding the medium. Thereafter, replace with fresh MIM-2 every 48 hours until the cell confluence reaches over 80%, approximately 10 days after EB seeding.

[0058] Pre-MSCs were passaged to P0: When the culture reached 80% confluence, the supernatant was discarded, and the cells were digested with TrypLE for 3-5 minutes. TBD complete medium was added to neutralize the digestion, and the cells were gently pipetted to form single cells. The cell suspension was collected for counting. RNA samples were collected from 1 million cells, and the remaining cells were passaged at 4 × 10⁻⁶ cells / year. 4 / cm 2 Inoculate into T75 culture flasks and culture in TBD complete medium;

[0059] After 24 hours, observe under a microscope and change the medium. When changing the medium, gently shake the culture plate to suspend the non-adherent cells, gently aspirate 3 / 4 of the supernatant (do not aspirate all of it), and add fresh culture medium. Thereafter, change the fresh TBD complete medium every 48 hours until the cells grow to 80% confluence or there are a large number of high-density areas, then digest and passage to P1. In this example, a large number of high-density areas appeared on day 14 of culture.

[0060] 2) hiPSC-MSC passage culture

[0061] When the culture reaches 80% confluence under a microscope, discard the supernatant, digest with TrypLE for 3-5 min, neutralize with TBD complete medium, gently pipette to separate into single cells, collect the cell suspension for counting, collect RNA samples from 1 million cells, and then process the remaining cells at a rate of 2 × 10⁻⁶ cells / year. 4 / cm 2 Inoculate into new T175 culture flasks with TBD complete medium, and then replace with fresh TBD medium every 48 h thereafter.

[0062] When P1 cells reach 80% confluence, they are digested and processed at a rate of 1×10⁻⁶. 4 / cm 2 Inoculate into a new T175 culture flask, using TBD complete medium, denoted as P2.

[0063] The subsequent passage steps are the same as in the previous paragraph, resulting in P5 generation cells, which are used for identification and subsequent experiments.

[0064] Example 2

[0065] This embodiment provides the results of the relevant experiments.

[0066] 1. Obtain a hiPSC clone (iPSC-WT) from UCBMC through reprogramming:

[0067] This invention uses human umbilical cord blood mononuclear cells as the starting somatic cells and establishes hiPSCs using the CTS Cytotune Sendai reprogramming kit. Figure 1 A in the image represents representative images (Scale bar = 200 μm) from passage 3 (iPSC-WT-P3) and passage 15 (iPSC-WT-P15) on day 20 after reprogramming. As can be seen, on day 20 after infection with the Sendai virus reprogramming vector, colonies with relatively large nucleoplasm and dense cell arrangement can be observed. These colonies are picked up with a syringe needle and seeded into a new culture plate. The cells are then passaged continuously to P3 by picking single clones, and typical pluripotent stem cell clones can be observed. The cells are then passaged to P15 to establish a stable monoclonal hiPSC-WT cell line.

[0068] 2. Clinical-grade hiPSC-WT possesses pluripotency and differentiation potential.

[0069] We characterized the pluripotency and differentiation capacity of P15 hiPSC-WT cells, including flow cytometry analysis of pluripotency markers and in vitro trigerm layer differentiation. Flow cytometry results are shown below. Figure 1 As shown in Figure B, the expression rates of the pluripotency markers NANOG, OCT4, SOX2, TRA-1-60, TRA-1-81, and SSEA4 are all above 97%.

[0070] In vitro, P15 hiPSC-WT cells were directly induced to differentiate into three germ layers using a kit via monolayer culture. Immunofluorescence staining was then used to detect germ layer-specific markers. Results are as follows: Figure 1 As shown in Figure C, cells differentiating into the endoderm are positive for the marker SOX17, cells differentiating into the mesoderm are positive for the marker BRACYURY, and cells differentiating into the ectoderm are positive for the marker β3-TUBULLIN. In summary, hiPSCs exhibit pluripotency and high purity, and can successfully differentiate into all three germ layers in vitro. This indicates that the hiPSC clones obtained from UCBMC through reprogramming in this invention possess normal pluripotency and differentiation capacity up to the 15th generation.

[0071] 3. Establishment of B2M gene knockout hiPSC monoclonal lines

[0072] Figure 2 In Example 1, step 4, after electroporation for 72 hours, cells were collected for first-generation sequencing to detect the knockout efficiency. It can be seen that the first-generation sequencing site starts from the 5' end, and the red arrows indicate the Cas9 cleavage sites corresponding to the sgRNA. A distinct peak formation occurs after the Cas9 cleavage sites, indicating the formation of a large number of indels in the mixed cells. The mixed cells were then cultured as single cells. After the single cells grew into small clones, 33 single clones were selected for first-generation sequencing verification. The results showed that clones 3, 18, and 22 were homozygous knockout clones of the B2M gene.

[0073] The first-generation sequencing profiles of the three monoclonal antibodies are as follows: Figure 2 As shown in B, C, and D, all three clones produced deletions at the Cas9 cleavage site that were not integer multiples of 3, indicating frameshift mutations. These results demonstrate that the B2M gene was successfully knocked out in the homozygous KO cell line. We selected the hiPSC-Bko-3 monoclonal cell line for further research.

[0074] 4. B2M knockout hiPSCs exhibit pluripotency, differentiation ability, and a normal karyotype.

[0075] A B2M knockout hiPSC cell bank was established by expanding hiPSC-Bko-3 cells three times, and their pluripotency, differentiation capacity, and genetic stability were evaluated. Figure 3 As shown, hiPSC-Bko-3 exhibits a typical clonal growth morphology of pluripotent stem cells. Figure 3 A in the sample), positive for alkaline phosphatase staining (A). Figure 3 (B in the original text), and has a normal chromosomal karyotype ( Figure 3 The immunofluorescence staining results of pluripotent markers showed that the hiPSC-Bko-3 cell line was positive for NANOG, TRA-1-81, SOX2, and OCT4. Figure 3 (D in the middle).

[0076] In vitro, hiPSC-Bko-3 cells were directly induced to differentiate into three germ layers using a kit via monolayer culture. Immunofluorescence staining was then used to detect germ layer-specific markers. Results are as follows: Figure 3 As shown in Figure E, cells differentiating towards the endoderm are positive for the marker SOX17, cells differentiating towards the mesoderm are positive for the marker BRACYURY, and cells differentiating towards the ectoderm are positive for the marker β3-TUBULLIN. These results indicate that the hiPSC-Bko-3 line with B2M gene knockout was successfully established.

[0077] 5. hUCMSCs were successfully isolated and cultured.

[0078] Primary hUCMSCs (denoted as P0) were isolated from human umbilical cord tissue using a mechanical fragmentation method. P0 hUCMSCs were then passaged to P5 for identification using enzymatic digestion. Surface markers were detected by flow cytometry. Results are as follows: Figure 4 As shown in Figure A, the expression rates of positive markers CD73, CD90, and CD105 are as high as 95% or more, while the expression of negative markers CD11, CD19, CD34, CD45, and HLA-DR is almost non-existent. We induced hUCMSC-P5 cells to differentiate into bone, cartilage, and adipocytes to detect the multi-lineage differentiation capacity of hUCMSCs, and obtained the following results: Figure 4 As shown in Figure B, hUCMSCs were stained with alizarin 28 days after osteogenic differentiation, revealing numerous red calcium crystals (left); stained with alizarin blue 18 days after chondrogenic differentiation, revealing blue-stained cartilage spheres (middle); and stained with Oil Red O 18 days after adipogenic differentiation, revealing red-stained lipid droplets (right). These results demonstrate the successful extraction of hUCMSCs from human umbilical cord tissue.

[0079] 6. Both hiPSC-WT and hiPSC-KO can differentiate into iMSCs efficiently.

[0080] To induce hiPSC differentiation into iMSCs, an embryoid body (EB) approach was employed, combining the principle of regulating germ layer development-related signaling pathways with small molecule compounds. The GSK3β inhibitor CHIR99021 was used to activate the WNT signaling pathway, while the TGFβ signaling pathway inhibitor SB431542 was used to induce hiPSC-WT and hiPSC-KO to differentiate into mesenchymal stem cells (iMSC-WT and iMSC-KO). BMP4 promoted iPSC differentiation into mesoderm, and L-Ascorbic acid promoted iPSC differentiation into mesoderm and iMSC proliferation. Figure 5 Figure A illustrates the differentiation induction protocol of this invention: EBs are suspended in hiPSC medium for 24 h using a cloning method to promote EB formation, then switched to MIM-1 induction medium, followed by MIM-2 induction medium after 3 days. On day 6, EBs are seeded onto the bottom surface of a culture plate for adherent culture to promote cell migration. Figure 5 As indicated by B, after approximately 10-14 days of culture, mesenchymal-like cells that have migrated from adherent EBs are harvested and seeded in TBD medium, designated as P0. Subsequently, following standard passage methods, when the confluence of iMSCs reaches 80%-90%, they are passaged continuously up to the P5 generation for quality evaluation and application. Figure 5C in the figure shows the typical morphologies of iMSC-WT and iMSC-KO before the P3 and P5 generations.

[0081] 7. iMSCs possess the typical surface biomarker expression characteristics and differentiation potential of MSCs.

[0082] Next, flow cytometry was used to analyze the expression of surface markers used for MSC cell type identification. The results are as follows: Figure 6 As shown, the proportion of the three positive biomarkers in iMSC-WT was as high as 95% or more, while the five negative biomarkers were almost not expressed. Figure 6 The results for iMSC-KO were similar (A). Figure 6 B).

[0083] The trilineage differentiation capacity of the two iMSCs (P5) was tested, and the results are as follows: Figure 7 As shown in Figure A, both iMSC-WT and iMSC-KO possess the differentiation capabilities of chondrogenic, adipogenic, and osteogenic processes.

[0084] 8. iMSCs possess similar cytokine secretion and immunosuppressive capabilities to UCMSCs.

[0085] The paracrine function and immunosuppressive capacity of MSCs are important indicators for evaluating MSC efficacy. Culture supernatants from three cell lines—UCMSCs, iMSC-WT (P5), and iMSC-KO (P5)—were collected, and the levels of four cytokines—LIF, HGF, KGF, and IL-1—in the culture supernatants were detected by ELISA. The results were as follows: Figure 7 As shown in Figure B, the levels of LIF, HGF, and IL-11 in the culture supernatants of the three cell types were similar, with no statistically significant differences. However, the levels of KGF in the culture supernatants of iMSC-WT and iMSC-KO were significantly higher than those in the UCMSC group. Studies have shown that hUCMSCs from different donor sources have different inhibitory abilities on BV2 cell proliferation, and the therapeutic effect of hUCMSCs on mouse spinal cord injury is positively correlated with their ability to inhibit BV2 cell proliferation in vitro. In this invention, BV2 cells were treated with conditioned medium from the three cell types, and the inhibitory effect on BV2 cell proliferation was observed. The results showed that the inhibitory ability of the three cell types on BV2 cell proliferation was approximately 80%, and there was no significant difference between the groups. Figure 7 (C).

[0086] 9. iMSCs have a normal karyotype.

[0087] Chromosomal abnormalities are one of the risk factors for tumorigenicity of cell therapy products. Karyotype analysis was performed on P5 generation iMSC-WT and iMSC-KO, and the results were as follows: Figure 8As shown in A, both types of iMSCs have normal 46XX female karyotypes.

[0088] 10. iMSCs are not tumorigenic.

[0089] Soft agar colony formation assay is a commonly used method for evaluating cell tumorigenicity in vitro. This invention used Fibroblast as a negative control and HeLa as a positive control to detect the soft agar colony formation of iMSC-WT (P5) and iMSC-KO (P5), obtaining the following results: Figure 8 As shown in Figure B, neither of the two cell types can form soft agar clones.

[0090] 11. Successful knockout of B2M reduced the stimulation of NK cells to release IFNγ.

[0091] First-generation sequencing was used to detect the knockout status of the B2M gene in iMSC-KO, and the results are as follows: Figure 9 As shown in Figures A and B, compared to iMSC-WT (P5), iMSC-KO (P5) has a 26-base deletion in exon 2 of the B2M gene (red box). The deletion location and number of bases are similar to those in iMSC-KO (P5). Figure 2 The B in the text is consistent.

[0092] The expression of B2M protein in three cell types was detected by immunofluorescence staining, and NESTIN was used for staining the cytoskeleton. The results are as follows: Figure 9 As shown in Figure C, no B2M positive staining was found in iMSC-KO (P5), confirming the absence of B2M expression in iMSC-KO (P5).

[0093] Next, the three cell types were inactivated with mitomycin C and then co-cultured with activated NK cells for 48 hours. The culture supernatant was collected and the IFNγ content was detected using ELISA. The results are as follows: Figure 10 As shown, unstimulated NK cells exhibited very low IFNγ release concentrations, all below 200 pg / mL. NK cells stimulated with iMSC-WT (P5) showed the highest IFNγ release concentration, reaching nearly 1000 pg / mL, significantly higher than the UCMSC and iMSC-KO (P5) groups. The IFNγ release concentration in NK cells stimulated with iMSC-KO was lower than that in UCMSC and iMSC-WT (P5), indicating that B2M knockout reduced the stimulation of NK cells with IFNγ secretion by iMSCs.

[0094] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0095] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for establishing induced pluripotent stem cells overcoming induction of immunogenicity, characterized by, The method comprises the following steps: a starting somatic cell is reprogrammed to establish an induced pluripotent stem cell line, and a single clone of the induced pluripotent stem cell line is obtained by continuous picking and subculturing, wherein the single clone of the induced pluripotent stem cell line is completely matched with the genotype of the parent cell; β2-microglobulin of the single clone of the induced pluripotent stem cell line is knocked out to obtain a single clone of the induced pluripotent stem cell with β2-microglobulin knocked out, i.e. an induced pluripotent stem cell overcoming immunogenicity.

2. The establishment method according to claim 1, characterized by, The starting somatic cell is umbilical cord blood mononuclear cell; and / or the reprogramming kit used is CTS Cytotune Sendai reprogramming kit; and / or the β2-microglobulin knockout is performed by using CRISPR-Cas9 technology.

3. The induced pluripotent stem cell overcoming immunogenicity obtained by the establishment method according to any one of claims 1-2.

4. The induced pluripotent stem cell that overcomes the induction of immunogenicity according to claim 3, characterized in that, The induced pluripotent stem cell overcoming immunogenicity has normal pluripotency and normal differentiation ability at the 15th generation or before.

5. A mesenchymal stem cell, characterized in that, The induced pluripotent stem cell overcoming immunogenicity is induced and differentiated according to any one of claims 3-4.

6. The mesenchymal stem cell of claim 5, wherein, The step of inducing and differentiating comprises: the induced pluripotent stem cell overcoming immunogenicity is subcultured and then placed in a directional differentiation medium for directional differentiation culture, and is subcultured to the first generation of differentiated cells; the first generation of differentiated cells is subcultured and cultured to the fifth generation to obtain the mesenchymal stem cells.

7. The mesenchymal stem cell of claim 6, wherein, The directional differentiation medium comprises the following components: Part 1: StemFit basic 03-A and StemFit basic 03-B are thawed at 2-8℃ overnight, mixed uniformly in a biological safety cabinet, and then divided into 50 mL centrifuge tubes; CHIR99021 is added to 3 μM, and SB431542 is added to 10 μM; Part 2: TBD medium additive is thawed at 2-8℃ overnight, and then the TBD basic medium and the TBD additive are mixed uniformly in a biological safety cabinet, and then divided into 50 mL centrifuge tubes; CHIR99021 is added to 3 μM, and SB431542 is added to 10 μM.

8. The mesenchymal stem cell of claim 7, wherein, After SB431542 is added to 10 μM, part 1 further comprises adding BMP4 to 50 ng / mL; after SB431542 is added to 10 μM, part 2 further comprises adding L-ascorbic acid to 100 μM.

9. The mesenchymal stem cell of claim 7, wherein, After the first part of the directional differentiation medium is added to form embryoid bodies in the system, the second part of the directional differentiation medium is added 3 days after the first part of the directional differentiation medium is added for culture.

10. The use of the induced pluripotent stem cell overcoming immunogenicity according to any one of claims 3-4 or the mesenchymal stem cell according to any one of claims 5-9 in the preparation of a drug for transplantation treatment.

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