Application of induced pluripotent stem cell-derived mesenchymal stem cells in preparation of medicine for treating acute hepatic failure

Abstract

The invention discloses application of induced pluripotent stem cell-derived mesenchymal stem cells in preparation of a medicine for treating acute hepatic failure, belongs to the technical field of biomedicine, and solves the problem of unsatisfactory treatment effect caused by immunological rejection when the mesenchymal stem cells are used for treating the acute hepatic failure in the prior art. The induced pluripotent stem cell-derived mesenchymal stem cell provided by the invention has a protective effect on acute liver injury, can improve the levels of aspartate aminotransferase and alanine aminotransferase, remarkably improves the liver function of the injured liver, reduces the level of inflammatory factor TNF-alpha, reduces the necrotic focus area, reduces inflammatory cell infiltration, and has the effects of preventing and treating acute liver injury. The apoptosis level of liver cells is reduced, and the level of CD4 + T cells is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of induced pluripotent stem cell-derived mesenchymal stem cells in the preparation of drugs for treating acute liver failure. Background Technology

[0002] The liver is the largest internal organ, playing vital roles such as producing bile, storing glycogen, and filtering harmful substances. Liver diseases, especially acute liver failure, pose a serious threat to lives worldwide. Acute liver failure (ALF) is a severe liver syndrome with an extremely high mortality rate, typically caused by multiple factors such as viral infections, alcohol, drugs, autoimmune disorders, and hepatotoxic substances, resulting in severe liver damage. Although liver transplantation remains an effective treatment for ALF, its application is severely limited due to severe organ shortages and immune rejection.

[0003] Previous studies have indicated that transplanted mesenchymal stem cells (MSCs) can improve liver function, inhibit hepatocyte apoptosis, and promote hepatocyte proliferation in animals with liver failure (ALF); in clinical trials, MSCs have also shown therapeutic effects in patients with liver failure. However, cell-mediated allogeneic immune rejection limits the persistence of MSCs in vivo, thus limiting their therapeutic potential. Summary of the Invention

[0004] This invention provides the application of induced pluripotent stem cell-derived mesenchymal stem cells in the preparation of drugs for treating acute liver failure, thereby solving the problem of unsatisfactory treatment effects caused by immune rejection when using mesenchymal stem cells to treat acute liver failure in the prior art.

[0005] This invention provides the application of induced pluripotent stem cell-derived mesenchymal stem cells in the preparation of drugs for treating acute liver failure.

[0006] In some embodiments, the method for establishing mesenchymal stem cells derived from induced pluripotent stem cells includes the following steps:

[0007] Induced pluripotent stem cell lines were established by reprogramming starting somatic cells, and monoclonal induced pluripotent stem cell lines were obtained by continuously selecting and passaged single clones.

[0008] The monoclonal induced pluripotent stem cell line was subjected to β2-microglobulin knockout to obtain β2-microglobulin knockout induced pluripotent stem cell monoclonals.

[0009] After the β2-microglobulin knockout induced pluripotent stem cell monoclonal revival and passage, the cells were placed in a directed differentiation medium for directed differentiation culture and passaged to the first generation of differentiated cells.

[0010] The first-generation differentiated cells were passaged and cultured to the fifth generation to obtain the mesenchymal stem cells.

[0011] In some embodiments, the initiating somatic cell is an umbilical cord blood mononuclear cell.

[0012] In this invention, umbilical cord blood mononuclear cells are the ideal donor cell type for producing hiPSCs.

[0013] In some implementations, the reprogramming kit used is the CTS Cytotune Sendai Reprogramming Kit;

[0014] And / or, the knockout of β2-microglobulin is performed using CRISPR-Cas9 technology.

[0015] In some embodiments, the directed differentiation culture medium comprises the following components:

[0016] Part 1: Thaw StemFit basic 03-A and StemFit basic 03-B overnight at 2-8℃, 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.

[0017] Part 2: Thaw the TBD medium supplement overnight at 2-8℃. Mix the TBD basal medium and TBD supplement thoroughly in a biosafety cabinet and dispense into 50 mL centrifuge tubes. Add CHIR99021 to 3 μM, SB431542 to 10 μM, and L-Ascorbic acid to 100 μM.

[0018] 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.

[0019] The inventors 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 hiPSCs into mesoderm and the properties of L-Ascorbic acid to promote the differentiation of hiPSCs into mesoderm and the proliferation of iMSCs, thereby achieving the directed induction of hiPSCs into mesenchymal stem cells (iMSCs).

[0020] In some embodiments, the dosage form of the drug is an injection.

[0021] In some embodiments, the drug uses the mesenchymal stem cells as the sole active ingredient.

[0022] In some embodiments, the drug is a drug used to improve the levels of aspartate aminotransferase and alanine aminotransferase.

[0023] In some embodiments, the drug is a drug used to reduce the level of the inflammatory factor TNF-α.

[0024] In some embodiments, the drug is a drug used to reduce the level of apoptosis in liver cells.

[0025] In some embodiments, the drug is a drug used to improve liver function.

[0026] In some embodiments, the drug is a drug used to reduce inflammatory cell infiltration in liver tissue.

[0027] In some embodiments, the drug is a drug used to reduce the area of ​​necrotic foci in liver tissue.

[0028] In some embodiments, the acute liver failure is caused by concanavalin A.

[0029] In some embodiments, the drug also includes pharmaceutically acceptable excipients.

[0030] This invention establishes a B2M gene knockout hiPSC (hiPSC-KO) lacking HLA-I class molecules, yet it retains pluripotency and genomic stability. This invention confirms the absence of B2M protein expression in hiPSC-KO and successfully induces hiPSC-KO to differentiate into iMSCs (iMSC-KO). 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.

[0031] The inventors' research indicates that the induced pluripotent stem cell-derived mesenchymal stem cells provided in this invention have a protective effect against acute liver injury. iMSC-KO significantly improves the survival rate of mice with liver injury, improves aspartate aminotransferase and alanine aminotransferase levels, significantly enhances liver function in damaged livers, reduces the level of the inflammatory factor TNF-α, reduces the area of ​​necrotic lesions, reduces inflammatory cell infiltration, reduces hepatocyte apoptosis, and reduces CD4+. +T cell levels. Transcriptome sequencing analysis showed that iMSC-KO treatment significantly reduced signaling pathways related to lymphocyte activation, differentiation, and proliferation, while increasing the activity of metabolic signaling pathways. Furthermore, compared to the iMSC-WT treatment group, the iMSC-KO treatment group showed significantly downregulated signaling pathways related to immune response and leukocyte chemotaxis, indicating that iMSC-KO possesses higher immune tolerance. Attached Figure Description

[0032] 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.

[0033] Figure 1 The process of obtaining hiPSC cloning from UCBMC through reprogramming is provided for embodiments of the present invention, wherein D20, P3, and P15 are representative hiPSC images (Scale bar = 200 μm) on day 20, generation 3, and generation 15, respectively.

[0034] Figure 2 The knockout efficiency and single-clone knockout verification of the B2M gene provided in the embodiments of the present invention, wherein, Figure 2 In the figure, A represents the sgRNA cleavage efficiency result provided in the embodiment of the present invention, wherein the black arrow indicates the direction of sgRNA and the red arrow indicates the cleavage site; Figure 2 B in the image represents the first-generation sequencing map of single clone #3. Figure 2 In the image, C represents the first-generation sequencing map of single clone #18; Figure 2 D in the sequence represents the first-generation sequencing map of single clone #22.

[0035] Figure 3 The phenotypic identification results of P5 generation UCMSCs provided in this embodiment of the invention, wherein, Figure 3 In this context, A represents the flow cytometry detection result of UCMSC surface markers; Figure 3 B in the image represents the staining results of the three lineages of differentiation. 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).

[0036] Figure 4 This invention provides an iMSC differentiation protocol and cell morphology at different differentiation stages, as illustrated in the embodiments of the present invention. Figure 4 In this context, A represents the iMSC differentiation protocol; Figure 4 B in the text represents the cell morphology observations on day 10 of iMSC differentiation, day 6 of P0, and day 3 of P3 generation. Figure 4 C in the figure represents the morphology of iMSC-WT and iMSC-KO in P3 and P5 generations (Scale bar = 200 μm).

[0037] Figure 5 The first-generation sequencing provided in this embodiment of the invention verifies the B2M gene knockout in iMSC-KO, wherein, Figure 5 In the diagram, A represents the first-generation sequencing data of the B2M gene locus in iMSC-WT. Figure 5 B in the diagram represents the first-generation sequencing data of the B2M gene locus of iMSC-KO. Figure 5 In this context, C indicates that iMSC-KO does not express B2M protein (Scale bar = 100 μm).

[0038] Figure 6 The ELISA analysis provided in this embodiment of the invention shows the differences in IFNγ release levels from NK cells stimulated by three different cell types, where n = 6. .

[0039] Figure 7 This is an experimental design diagram for stem cell therapy in ConA-induced ALF mice, provided in an embodiment of the present invention.

[0040] Figure 8 Survival curves of ConA-induced ALF mice provided in this embodiment of the invention (preliminary experiment, n=10).

[0041] Figure 9 Survival curves of ConA-induced ALF mice with different cell therapies provided in embodiments of the present invention, n = 10.

[0042] Figure 10 The peripheral blood plasma ALT and AST levels of mice at 12 and 24 hours provided in this embodiment of the invention are as follows: Figure 10 In this context, A represents the peripheral blood ALT levels of mice in the 12h and 24h groups. n = 6; Figure 10 In the figure, B represents the peripheral blood ALT levels of mice in the 12h and 24h groups. , n = 6; KU represents the Kamen unit.

[0043] Figure 11 The TNF-α levels in mouse peripheral blood plasma at 12 and 24 hours provided in this embodiment of the invention; .

[0044] Figure 12HE staining of mouse liver tissue sections at 12h and 24h provided in this embodiment of the invention. Blue arrows indicate necrotic foci, and orange arrows indicate inflammatory cell infiltration. Scale bar = 200 μm.

[0045] Figure 13 The 12h and 24h liver cleaned caspase 3 levels in each group of mice provided in the embodiments of the present invention; wherein, Figure 13 In the figure, A represents the Western blot band plot; Figure 13 In the figure, B represents the bar chart obtained by standardizing the gray values ​​of the Cleaved caspase3 protein band using β-actin as an internal reference. .

[0046] Figure 14 The liver pathological sections of mice in each group provided in the embodiments of the present invention were stained with Bcl2 at 12h and 24h after treatment, with a scale bar of 200 μm.

[0047] Figure 15 Principal component clustering (PCA) of five mouse PBMC transcriptomes provided in this embodiment of the invention, n=3.

[0048] Figure 16 This invention provides differential gene and functional enrichment analysis between ConA mice and control mice in embodiments of the invention; wherein, Figure 16 In the diagram, A represents the differential gene volcano plot of the PBMC transcriptome between the two groups of mice; Figure 16 In the diagram, B represents the heatmap of the top 25 upregulated and downregulated genes, ALF represents the ALF model group, and Sham represents the control group. Figure 16 C in the figure represents the GO-BP enrichment results of upregulated and downregulated genes; Figure 16 D in the figure represents the KEGG enrichment results of upregulated and downregulated genes.

[0049] Figure 17 This invention provides a differential gene and functional enrichment analysis between ConA+iMSC-KO mice and ALF model mice in an embodiment of the invention; wherein, Figure 17 In the diagram, A represents the differential gene volcano plot of the PBMC transcriptome between the two groups of mice; Figure 17 B in the figure represents the GO-BP enrichment results of upregulated and downregulated genes; Figure 17 C in the figure represents the KEGG enrichment results of upregulated and downregulated genes.

[0050] Figure 18 CD4 immunohistochemical staining of tissue sections from each group of mice provided in the embodiments of the present invention at 12h and 24h after treatment, with a scale bar of 200 μm.

[0051] Figure 19This invention provides a differential gene and functional enrichment analysis between ConA+iMSC-KO mice and ConA+UCMSC mice in embodiments of the invention; wherein, Figure 19 In the diagram, A represents the differential gene volcano plot between the two groups of mice; Figure 19 B in the graph represents the differentially expressed gene GO enrichment map; Figure 19 C in the diagram represents the differential gene KEGG enrichment map.

[0052] Figure 20 This invention provides a differential gene and functional enrichment analysis between ConA+iMSC-KO mice and ConA+iMSC-WT mice in an embodiment of the invention; wherein, Figure 20 In the diagram, A represents the differential gene volcano plot; Figure 20 B in the graph represents a differential gene heatmap; Figure 20 C in the figure represents GO enrichment analysis; Figure 20 D in the figure represents KEGG enrichment analysis. Detailed Implementation

[0053] 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.

[0054] To address the problem of unsatisfactory treatment effects caused by immune rejection when using mesenchymal stem cells to treat acute liver failure in existing technologies, this embodiment provides an application of induced pluripotent stem cell-derived mesenchymal stem cells in the preparation of drugs for treating acute liver failure.

[0055] In this invention, UCMSCs, iMSC-WT, and iMSC-KO were used in a mouse model of acute liver injury induced by concanavalin A (ConA). Results showed that the survival rate of mice in the iMSC-KO treatment group was controlled, while almost all mice in the other groups died within 48 hours. This invention also found that in ALF mice treated with iMSC-KO, the levels of peripheral blood aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly improved at 12 and 24 hours, and the level of tumor necrosis factor-α (TNF-α) was also significantly reduced at 12 hours. Histological examination also showed that iMSCs… Mice treated with iMSC-KO showed smaller necrotic lesions in their livers and the lowest levels of inflammatory cell infiltration among all groups. Furthermore, this invention analyzed the Bcl2 and Cleaved-caspase 3 protein levels in the liver tissues of mice in each group. The results showed that the iMSC-KO treatment group had the lowest Bcl2 protein content in its liver, and the Cleaved-caspase 3 protein content was the lowest among all ALF groups. These results indicate that iMSC-KO treatment has a protective effect against ConA-induced acute liver injury, significantly improves liver function in ALF mice, reduces the area of ​​necrotic lesions, and decreases inflammatory cell infiltration and hepatocyte apoptosis.

[0056] To further investigate the mechanism by which iMSC-KO cells counteract ConA-induced acute liver injury, this invention evaluated CD4 levels in different liver groups. + The level of T cells. It has been reported that CD4... + T cells have been shown to increase ConA-induced acute liver injury. In the study of this invention, iMSC-KO cells significantly reduced CD4+ in the liver. + The level of T cells. Mouse peripheral blood transcriptome sequencing results also showed that, compared with the ALF model group, ALF mice treated with iMSC-KO had significantly downregulated signaling pathways related to lymphocyte proliferation, differentiation, and activation, while significantly increased levels of metabolic-related signals. These results indicate that iMSC-KO cells can regulate CD4+. + T cells are used to suppress ConA-induced acute liver injury.

[0057] This invention also compared the differences in peripheral blood transcriptomes between two groups of mice treated with iMSC-KO and iMSC-WT. The results showed that iMSC-KO treatment significantly reduced ConA-induced immune and inflammation-related signaling pathways, and weakened leukocyte chemotaxis and migration. This difference in tolerance to the immune response may be related to the loss of HLA-I molecules due to B2M gene knockout, thereby reducing immune rejection.

[0058] The C57BL / 6N strain mice used in these embodiments of the invention were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed at the Animal Experiment Center of Tongji University. All mice were housed in an air-conditioned environment, experiencing periodic light-dark cycles every 12 hours, and were provided with standard pelleted feed, while having free access to food and water. All animal experiments were conducted in accordance with animal welfare and ethical requirements.

[0059] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0060] Example 1

[0061] This embodiment provides an experiment for preparing mesenchymal stem cells derived from induced pluripotent stem cells.

[0062] I. Experimental Methods

[0063] 1. Isolation and culture of umbilical cord blood mononuclear cells:

[0064] UCBMC medium (prepared fresh, after mixing StemPro™-34 medium and StemPro™-34 Nutrient supplement, 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. Human umbilical cord blood mononuclear cells (UCBMCs) were removed from liquid nitrogen and rapidly thawed in a water bath. The thawed UCBMCs were slowly added along the wall of the centrifuge tube to the 15 mL centrifuge tube containing the aforementioned UCBMC medium. The mixture was gently shaken and centrifuged at 1500 rpm for 5 minutes at room temperature. min; discard the supernatant after centrifugation, add UCBMC medium, mix well to resuspend the cells, and obtain a cell suspension; transfer the cell suspension to a T25 cell culture flask, add medium to 5 mL, and induce culture at 37℃ and 5% CO2 for 3 days; during induction, observe the cells and add fresh UCBMC medium, i.e., gently aspirate 2 mL of medium from each flask and add 2 mL of fresh medium; if cells are present during aspiration, centrifuge at 1500 rpm for 5 min at room temperature, discard the supernatant, resuspend the cells with 2 mL of fresh medium, and then add the cells back to the culture system.

[0065] 2. Infection by Sendai virus reprogramming vector:

[0066] 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. 6 Cell 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.

[0067] 3. Single-clone selection and subculturing:

[0068] 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 the degree of colony differentiation increases. Then, perform cell passage to obtain the monoclonal induced pluripotent stem cell line hiPSC-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 0.2 mL of E8 complete medium in each well. Discard the cell culture medium in the 48-well plate with mLE8 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.

[0069] 4. B2M gene knockout was used to establish a lineage, resulting in a single clone of induced pluripotent stem cells (hiPSC-KO) with β2-microglobulin knockout:

[0070] 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 hiPSC-WT cells were digested with the dissociative enzyme Accutase to form single cells 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, the pellet was collected. One portion was cryopreserved 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 and cultured for 4-5 days. The cells were observed to grow into small clones without contact with each other. Under a microscope, a single clone with good morphology was picked with a 10 μL pipette tip, pipetted three 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 collected 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.

[0071] 5. hiPSC culture and induced differentiation

[0072] 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&D systems;

[0073] 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.

[0074] P18 passage hiPSC cells were revived, and the passage number after revival was designated as P19. The cells were passaged twice consecutively until P21, yielding P21 passage hiPSC cells.

[0075] 1) Directed differentiation of hiPSC-MSC:

[0076] D-1: When P21 generation hiPSC cells reached 80% confluence, they were digested using ReleSR, and the hiPSC cell clumps were resuspended in StemFit basic 03 complete medium, with Y27632 added to 10 μM. The cell clump suspension was transferred to a low-attachment 6 cm bacterial culture dish and cultured in suspension.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] D4~D5: Change MIM-2 daily. When changing the solution, you can directly aspirate the supernatant, but be careful not to aspirate the EB.

[0081] 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℃.

[0082] 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.

[0083] When the culture reaches 80% confluence, 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 store the remaining cells at 4 × 10⁻⁶ cells per cell line. 4 / cm 2 Inoculate into T75 culture flasks and culture in TBD complete medium;

[0084] 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 there are a large number of high-density areas. Digest and passage to the first generation (P1 generation) to obtain P1 generation differentiated cells. In this example, a large number of high-density areas appeared on day 14 of culture.

[0085] 2) hiPSC-iMSC passage culture

[0086] When the culture reaches 80% confluence under a microscope, discard the supernatant, digest with TrypLE for 3-5 min, then digest again with TBD complete medium, gently pipette to separate into single cells, collect the cell suspension for counting, and collect RNA samples from 1 million cells. The remaining cells are then processed at a rate of 2 × 10⁻⁶ cells / year. 4 / cm 2 The cells were seeded into new T175 culture flasks in TBD complete medium, which was then replaced with fresh TBD medium every 48 hours. When the P1 generation differentiated cells reached 80% confluence, they were digested and cultured at a concentration of 1×10⁻⁶ cells / mL. 4 / cm 2 Inoculate into new T175 culture flasks, using TBD complete medium, and designate as the second generation (P2 generation).

[0087] The subsequent passage steps are the same as the previous paragraph, and the cells are cultured to the fifth generation (P5 generation) for identification and subsequent experiments.

[0088] II. Experimental Results

[0089] 1. Obtain a hiPSC clone (hiPSC-WT) from UCBMC through reprogramming:

[0090] This invention uses human umbilical cord blood mononuclear cells (UCBMCs) as the starting somatic cells and establishes hiPSCs using the CTS CytotuneSendai reprogramming kit. Figure 1 Representative images (Scale bar = 200 μm) of the 3rd generation (P3 generation hiPSC-WT) and the 15th generation (P15 generation hiPSC-WT) on day 20 after reprogramming show that, 20 days after infection with the Sendai virus reprogramming vector (D20), colonies with relatively large nucleoplasm and dense cell arrangement can be observed. These colonies were picked with a syringe needle and seeded into a new culture plate. The cells were then passaged continuously to the P3 generation (P3) by picking single clones, and typical pluripotent stem cell clones could be observed. The cells were then passaged to the P15 generation (P15) to establish a stable monoclonal hiPSC-WT cell line (i.e., P15 generation hiPSC-WT) for subsequent research.

[0091] 2. Establishment of hiPSC monoclonal lines by knocking out the B2M gene (β2 microglobulin gene).

[0092] Figure 2 In the above experimental method, A represents cells collected after electroporation for 72 hours in step 4 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 arrow indicates the Cas9 cleavage site corresponding to the sgRNA. A distinct peak formation occurs after the Cas9 cleavage site, indicating the formation of a large number of indels in the mixed cells. The mixed cells were 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 (denoted as ANB-Bko2-3, ANB-Bko2-18, and ANB-Bko2-22, respectively) were homozygous knockout clones of the B2M gene. The first-generation sequencing maps of the three single clones are shown below. Figure 2 As shown in B, C, and D, all three clones produced deletions that were not multiples of 3 at the Cas9 cleavage site, resulting in frameshift mutations. These results indicate that the B2M gene was successfully knocked out in the homozygous KO cell line. In this invention, the ANB-Bko2-3 monoclonal cell line (i.e., hiPSC-KO) was selected for further research.

[0093] 3. UCMSCs were successfully isolated and cultured.

[0094] Primary umbilical cord mesenchymal stem cells (P0 generation UCMSCs) were isolated from human umbilical cord tissue using a mechanical fragmentation method. The P0 generation UCMSCs were passaged to the P5 generation using enzymatic digestion to obtain P5 generation UCMSCs. The P5 generation UCMSCs were identified, and surface markers were detected by flow cytometry. The results are as follows: Figure 3 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. By inducing P5 generation UCMSCs to differentiate into bone, cartilage, and adipocytes, the multi-lineage differentiation capacity of UCMSCs was tested, and the results were as follows: Figure 3 As shown in Figure B, after 28 days of osteogenic differentiation, UCMSCs stained with alizarin showed abundant red calcium crystals (left); after 18 days of chondrogenic differentiation, alizarin blue staining revealed blue-stained cartilage spheres (middle); and after 18 days of adipogenic differentiation, Oil Red O staining revealed red-stained lipid droplets (right). These results demonstrate the successful acquisition of umbilical cord mesenchymal stem cells (UCMSCs) from human umbilical cord tissue, and subsequent experiments were conducted using P5 generation UCMSCs.

[0095] 4. Both hiPSC-WT and hiPSC-KO can differentiate into iMSCs efficiently.

[0096] To induce hiPSCs to differentiate into iMSCs, an embryoid body (EB) approach was used, combining the principle of small molecule compounds regulating germ layer development-related signaling pathways. 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 (i.e., P15 generation hiPSC-WT) and hiPSC-KO (i.e., ANB-Bko2-3) to differentiate into mesenchymal cell-like cells (iMSC-WT and iMSC-KO). BMP4 can promote the differentiation of hiPSCs into the mesoderm, and L-Ascorbic acid can promote the differentiation of hiPSCs into the mesoderm and the proliferation of iMSCs. Figure 4 Figure A illustrates the differentiation induction protocol of this invention: EBs are suspended in hiPSC medium for 24 hours using a clonate splitting 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 4As shown in B, after about 10 days of culture, mesenchymal-like cells that have crawled out of the adherent EBs are harvested and seeded in TBD medium, denoted as P0; then, following the conventional passage method, when the confluence of iMSCs reaches 80%~90%, they are passaged, and the culture is continuously passaged until the P5 generation. Figure 4 Figure C shows the typical morphologies of iMSC-WT and iMSC-KO in the P3 and P5 generations, respectively. P5 generation iMSC-WT and P5 generation iMSC-KO were used for subsequent experiments.

[0097] 5. Successful knockout of the B2M gene weakened the stimulation of NK cells to release IFNγ.

[0098] First-generation sequencing was used to detect the knockout of the B2M gene in P5 generation iMSC-KO, and the results are as follows: Figure 5 As shown in Figures A and B, compared to P5 generation iMSC-WT, the P5 generation iMSC-KO 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 Figures A and B. Figure 2 The expression of B2M protein in the three cell types was detected using immunofluorescence staining, with NESTIN used for cytoskeleton staining. The results are as follows: Figure 5 As shown in C, no B2M positive staining was found in P5 generation iMSC-KO, confirming the absence of B2M protein expression in P5 generation iMSC-KO.

[0099] Next, the three cell types (P5 generation UCMSCs, P5 generation iMSC-WT, and P5 generation iMSC-KO) were inactivated with mitomycin C and then co-cultured with activated NK cells for 48 h. The culture supernatant was collected and the IFNγ content was detected by ELISA. The results are as follows: Figure 6 As shown, unstimulated NK cells (NK) exhibited very low IFNγ release concentrations, all below 200 pg / mL. NK cells stimulated with P5 generation iMSC-WT (NK+iMSC-WT) showed the highest IFNγ release concentration, reaching nearly 1000 pg / mL, significantly higher than NK cells stimulated with UCMSCs (NK+UCMSC) and NK cells stimulated with P5 generation iMSC-KO (NK+iMSC-KO). NK cells stimulated with P5 generation iMSC-KO (NK+iMSC-KO) showed lower IFNγ release concentrations than NK cells stimulated with UCMSCs (NK+UCMSC) and NK cells stimulated with P5 generation iMSC-WT (NK+iMSC-WT), indicating that B2M gene knockout reduced the stimulation of NK cells by iMSCs to secrete IFNγ.

[0100] Example 2

[0101] This embodiment provides an application experiment of mesenchymal stem cells derived from induced pluripotent stem cells.

[0102] The iMSC-WT (i.e., P5 generation iMSC-WT), iMSC-KO (i.e., P5 generation iMSC-KO), and umbilical cord mesenchymal stem cells (i.e., P5 generation UCMSC) used in this embodiment all came from Example 1.

[0103] Experimental grouping and procedures are as follows Figure 7 As shown, the specific steps were as follows: 100 mg of concanavalin A (ConA) was dissolved in 16 mL of sterile physiological saline to prepare a working solution of 6.25 mg / mL. The solution was aliquoted into 1 mL vials and stored at -20℃. One hundred and ten 6-week-old male C57BL / 6N healthy mice were used, and acclimatized for one week before the experiment. The mice were randomly divided into five groups of 22 each, numbered according to cage number and ear tag. The treatments for the five groups are shown in Table 1.

[0104] Table 1. Treatment status of 5 groups

[0105]

[0106] Mice were restrained with a fixator, exposing their tails. The ventral side of the tail was wiped with an alcohol swab to expose the tail vein. Following the groupings in Table 1, ConA or cell suspension was drawn up with a 1 mL syringe and slowly injected into the mice via the tail vein. The injection time was recorded. Ten mice were selected from each group, and their survival was observed at 6h, 12h, 24h, 48h, 72h, 96h, and 120h after treatment. Survival curves were plotted.

[0107] This embodiment also provides the results of related experiments.

[0108] 1. A ConA-induced mouse model of acute liver failure was successfully established.

[0109] Before conducting formal experiments, this invention performed a preliminary experiment to detect whether ConA induces acute liver failure in mice. Twenty C57BL / 6N mice were divided into two groups, with one group injected with the working solution (ConA, 25 mg / kg). The survival status of the mice was observed, and the results are as follows: Figure 8 As shown, during the 72-hour observation period, no mice in the control group died, while in the ALF model group, one mouse died 8 hours after ConA injection, followed by one mouse at 11 hours, two at 26 hours, one at 28 hours, three at 45 hours, and one at 60 hours, with only one mouse surviving by 72 hours. This indicates that ConA can effectively induce acute liver failure in C57BL / 6N mice within a short period, leading to death.

[0110] 2. Differences in the improvement of ALF mouse survival rate among the three cell groups.

[0111] According to Table 1 and Figure 7 The experimental design was used to create a model and administer stem cell therapy. The survival of mice was observed over 120 hours, and the results were as follows: Figure 9 As shown, no mice in the control group died during the 120-hour observation period; however, in the ALF model group, the mortality rate reached 90% at hour 10 and all mice died by hour 30; in the ConA+UCMSC group, mice also died successively after treatment, reaching 90% by hour 20, with the remaining 10% of mice surviving in subsequent observations; in the ConA+iMSC-WT group, 70% of mice died by hour 20, with the remaining 30% surviving until hour 50, all of whom died; in the ConA+iMSC-KO group, 60% of mice died by hour 20, with the remaining 40% surviving to the end of the 120-hour observation period without any further deaths. Survival analysis results indicate that all three cell therapies improved mouse survival rates, with statistically significant differences compared to ConA. Notably, ALF mice treated with iMSC-KO showed the highest survival rate.

[0112] 3. Differences in the effects of three cell groups on the liver biochemical indicators aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in ALF mice.

[0113] This invention extracted peripheral blood plasma from mice 12 hours and 24 hours after treatment and used a microplate biochemical analysis to detect the levels of ALT and AST in the plasma. The results are as follows: Figure 10As shown, compared with the control group, ALT and AST levels in all treatment groups were significantly elevated at 12h and 24h, confirming the liver damage caused by ConA treatment in mice. For 12h plasma, ALT and AST levels decreased in all three stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group), with the ALT decrease in the ConA+iMSC-KO group being statistically significant. The decrease in AST levels in all three stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group) was statistically significant, and the decrease in AST levels in the two iMSC treatment groups (ConA+iMSC-WT group and ConA+iMSC-KO group) was also statistically significant compared to the ConA+UCMSC group. For 24-hour plasma, compared with the ALF model group, ALT levels decreased in all three stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group), with the decrease in plasma ALT in the ConA+iMSC-KO group being statistically significant. However, AST levels did not decrease significantly in any of the three stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group). These results indicate that ConA treatment caused substantial liver damage in mice, and different cell therapies improved liver function during the acute injury phase (12 hours), with iMSC-KO treatment showing a more significant improvement. As the injury time prolonged, iMSC-KO showed a sustained effect in improving liver function, while the other two cell groups (UCMSC and iMSC-WT) did not show significant improvement.

[0114] 4. Effects of three cell groups on the inflammatory cytokine TNF-α in peripheral blood of ALF mice

[0115] Next, the changes in the inflammatory factor TNF-α in peripheral blood were analyzed using ELISA. The results are as follows: Figure 11 The results showed that 12 hours after treatment, the TNF-α level in the ALF model group mice was significantly increased, and the difference was statistically significant. Compared with the ALF model group, the TNF-α level in the peripheral blood of mice in the three stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group) was significantly decreased, and the difference was statistically significant. There was no statistically significant difference among the three stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group). 24 hours after treatment, the TNF-α level in the peripheral blood of mice in all groups decreased to normal levels.

[0116] 5. iMSC-KO significantly reduced inflammatory cell infiltration and necrotic foci in the liver tissue of ALF mice.

[0117] Liver tissues were collected from mice in each group 12 h and 24 h after treatment. After fixation, paraffin embedding, sectioning, and HE staining, the degree of inflammatory cell infiltration and tissue necrosis in the liver tissues of each group of mice after treatment was observed. Figure 12 The results show that, compared with the control group, all treatment groups (ALF model group, ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group) exhibited varying degrees of tissue necrosis, characterized by lighter eosin staining, shrunken or lysed cell nuclei, and incomplete nuclear membranes (blue arrows). Extensive bleeding was also observed in the interstitial spaces, indicating that ConA treatment induced hepatocyte death and tissue congestion. The area of ​​necrotic foci in the liver of the ALF model group was significantly higher than that of the stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group) 12 hours after treatment, with large areas of necrotic whitening visible at 24 hours. In contrast, the area and level of necrotic foci in the liver tissue of the stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group) were reduced to varying degrees, especially in the ConA+iMSC-KO group. Inflammatory cell infiltration was observed around the portal vein in the livers of mice in all treatment groups (ALF model group, ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group), with the highest infiltration rate in the ALF model group. Among the three cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group), the ConA+iMSC-WT group showed the highest level of inflammatory cell infiltration around the portal vein in the livers, which may be related to graft immune rejection.

[0118] 6. iMSC-KO transplantation significantly reduced apoptosis levels in ALF mouse hepatocytes.

[0119] This invention used Western blot hybridization to compare the content of Cleaved-caspase 3 protein in the liver tissue of mice in different groups. The protein bands are shown in the figure. Figure 13 As shown in Figure A; the gray values ​​of the protein bands were analyzed using ImageJ software. After standardization with the gray values ​​of the internal reference protein, a statistical chart of the relative protein content was plotted, and the results are shown in Figure A. Figure 13As shown in B, compared with the control group, the Cleaved-caspase 3 protein content in the liver of mice in each treatment group (ALF model group, ConA+UCMSC group, ConA+iMSC-WT group, ConA+iMSC-KO group) increased to varying degrees, with the increase in Cleaved-caspase 3 protein content in the ALF model group being significant. Compared with the ALF model group, the Cleaved-caspase 3 protein content in mice in each stem cell therapy group (ConA+UCMSC group, ConA+iMSC-WT group, ConA+iMSC-KO group) decreased, with the decrease in Cleaved-caspase 3 protein content in the ConA+iMSC-KO group being statistically significant.

[0120] Immunohistochemistry was further used to detect the expression of Bcl2 protein in the livers of mice in each group. The results are as follows: Figure 14 As shown, almost no Bcl2-positive areas were found in the livers of control mice. However, in the livers of mice in all treatment groups (ALF model group, ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group), a large number of Bcl2-positive cells were found around the portal vein and in the interstitial spaces (blue arrows), with the ALF model group showing the highest concentration, and the entire necrotic lesion exhibiting positive staining. Among the three stem cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group), the ConA+iMSC-KO group had the fewest Bcl2-positive cells (the decrease in Bcl2 protein content was the most significant), indicating that iMSC-KO treatment had the best effect on improving apoptosis in mouse liver tissue.

[0121] 7. Effects of three cell groups on the transcriptome of peripheral blood mononuclear cells in ALF mice

[0122] After confirming the ameliorative effects of three stem cell therapies (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group) on liver function in ALF mice, peripheral blood mononuclear cells (PBMCs, 3 samples from each group, totaling 15 samples) with the most significant improvement at 12 hours were selected for transcriptome sequencing analysis. The raw sequencing data, after filtering, alignment to a reference genome, and calculation of FPKM values, underwent principal component analysis (PCA) on the 15 samples. The results are as follows: Figure 15As shown in the PCA diagram, there is a significant distance between the three control group samples and the other 12 samples. Among the four groups treated with ConA, the ConA+iMSC-WT group and the ConA+iMSC-KO group are relatively far from the ALF model group, while the ConA+UCMSC group shows a high degree of similarity to the ALF model group. The PCA results indicate that ConA treatment has a significant impact on the mouse PBMC transcriptome, and the mice treated with iMSC-WT and iMSC-KO show even lower similarity to the PBMC transcriptome of the ALF model group.

[0123] 8. ConA treatment significantly activated peripheral blood lymphocyte responses in mice.

[0124] Next, through gene differential analysis, GO and KEGG enrichment analysis, differentially expressed genes and differentially expressed signaling pathways in the PBMC transcriptomes of ALF model group and control group mice were compared, and the results were as follows: Figure 16 The results are shown. (By...) Figure 16 As shown in A, using |logFC| > 1 and P-value < 0.05 as the boundary, a volcano plot was drawn, revealing 1789 upregulated genes and 1849 downregulated genes. The genes were sorted according to |logFC|, and the 25 upregulated and 25 downregulated genes with the most significant differences were selected to create a heatmap. Figure 16 As shown in B, a large number of chemokine genes, such as Ccl12, Ccl24, Ccl7, and Cxcl9, were upregulated in the ALF model group. Inflammation-related genes, such as matrix metalloproteinase Timp1 and myeloperoxidase Mpo, were also significantly upregulated. GO and KEGG enrichment analyses were performed on the differentially expressed genes. The GO enrichment results are shown below. Figure 16 As shown in C, within the biological processes (BF) ontology of GO, the PBMC transcriptome of ALF model mice showed significant upregulation of signaling pathways such as immune response, lymphocyte differentiation, B cell activation, T cell activation, granulocyte proliferation, and lymphocyte proliferation, while genes related to mitosis and cell cycle were significantly downregulated; KEGG enrichment results are shown in... Figure 16 As shown in D, signaling pathways such as hematopoietic cell lineage, platelet activation, Th17 cell differentiation, Th1 and Th2 cell differentiation, and T cell receptor signaling were significantly upregulated, while signaling pathways such as cell cycle and DNA replication were significantly downregulated. These results indicate that lymphocyte responses and immune responses are highly activated in the peripheral blood of ConA-induced ALF mice.

[0125] 9. iMSC-KO significantly attenuated peripheral blood lymphocyte activation and enhanced metabolic function in mice.

[0126] Next, this invention compared differentially expressed genes and differentially expressed signaling pathways in the PBMC transcriptomes of mice in the ConA+iMSC-KO group and the ALF model group. The results are as follows: Figure 17 As shown, by Figure 17 As shown in A, using |logFC|>1 and Pvalue<0.05 as the boundary, a volcano plot was drawn, revealing 1045 upregulated genes and 549 downregulated genes. GO and KEGG enrichment analyses were performed on the differentially expressed genes. The GO enrichment results are shown below. Figure 17 As shown in Figure B, in the biological process BF ontology of GO, the PBMC transcriptome of ConA+iMSC-KO group mice showed significant activation of signaling pathways related to metabolism and synthesis (small molecule metabolism, organic acid metabolism, carboxylic acid metabolism, fatty acid metabolism, fatty acid synthesis), lipid transport, etc., while signaling pathways such as lymphocyte differentiation, T cell differentiation, lymphocyte activation, and T cell activation were significantly downregulated. KEGG enrichment results are shown below. Figure 17 As shown in Figure C, signaling pathways such as retinol metabolism, drug metabolism, steroid hormone synthesis, carbon metabolism, and amino acid synthesis were significantly upregulated, while signaling pathways such as Th17 cell (helper T cell 17, a newly discovered subset of T cells that secrete interleukin 17 (IL-17)) differentiation, T cell receptor signaling, and Th1 and Th2 cell differentiation were significantly downregulated. These results indicate that in ALF mice treated with iMSC-KO, liver metabolic and synthetic functions were significantly enhanced, and lymphocyte activation levels, especially T cell activation, were significantly improved.

[0127] To observe the aggregation and activation of lymphocytes in mouse liver tissue, immunohistochemistry was used to detect the expression of CD4 in the liver of each group of mice. The results are as follows: Figure 18 As shown. CD4 was almost never found in the livers of control mice. + T cells were observed in the periportal vein and interstitial spaces of the livers of mice in all treatment groups (ALF model group, ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group). + T cells (blue arrows) were most abundant in the ALF model group. Among the three cell therapy groups (ConA+UCMSC group, ConA+iMSC-WT group, and ConA+iMSC-KO group), the ConA+iMSC-KO group showed the highest CD4 count. + T cells were the least abundant, while more CD4 cells were found in the livers of mice in the ConA+iMSC-WT group. + T cells, which may be related to the high expression level of HLA-I molecules in iMSC-WT, leading to immune rejection.

[0128] 10. iMSC-KO treatment showed superior improvement in metabolic function in ALF mice compared to the UCMSC treatment group.

[0129] exist Figure 15 The PCA results revealed differences between the ConA+iMSC-KO and ConA+UCMSC groups. Subsequently, differentially expressed genes and signaling pathways in the PBMC transcriptomes of the two mouse groups were compared. The results are as follows... Figure 19 As shown, by Figure 19 As shown in A, using |logFC|>1 and Pvalue<0.05 as the boundary, a volcano plot was drawn, revealing 630 upregulated genes and 500 downregulated genes. GO and KEGG enrichment analyses were performed on the differentially expressed genes. The GO enrichment results are shown below. Figure 19 As shown in Figure B, it can be seen that in the GO BF ontology, the PBMC transcriptome of ConA+iMSC-KO group mice showed significant activation of signaling pathways related to metabolism and synthesis (small molecule metabolism, organic acid metabolism, carboxylic acid metabolism, fatty acid metabolism, fatty acid synthesis), lipid transport, etc. KEGG enrichment results are shown below. Figure 19 As shown in Figure C, signaling pathways such as retinol metabolism, drug metabolism, steroid hormone synthesis, carbon metabolism, and amino acid synthesis were significantly upregulated, while signaling pathways such as NK cell-mediated cytotoxicity and neutrophil extracellular trap formation were significantly downregulated. These results indicate that ConA+iMSC-KO mice exhibit strong metabolic function, demonstrating good liver function, and show some tolerance to NK cell killing.

[0130] 11. Immune response-related signaling pathways were significantly downregulated in the ConA+iMSC-KO group.

[0131] Next, we compared the differentially expressed genes and signaling pathways in the PBMC transcriptomes of the ConA+iMSC-KO and ConA+iMSC-WT mouse groups. The results are as follows: Figure 20 As shown, by Figure 20 As shown in A, using |logFC|>1 and Pvalue<0.05 as the boundary, a volcano plot was drawn, revealing 114 upregulated genes and 165 downregulated genes. Among these, the upregulated genes in ConA+iMSC-KO were mainly histone-related genes, while the downregulated genes primarily included Tlr3, Cxcl1, Ccl17, Ccl2, Cxcl3, Cxcl12, and Il1b, which are related to T cell responses, leukocyte chemotaxis, and inflammation. Figure 20 (B in the text). GO and KEGG enrichment analyses were performed on the differentially expressed genes. The GO enrichment results are shown below. Figure 20As shown in C, in the GO BF ontology, the nucleosome signaling pathway was upregulated in the PBMC transcriptome of ConA+iMSC-KO mice, while the activity of immune and inflammation-related signaling pathways, such as cytokine activity, NF-κB signaling, leukocyte chemotaxis, and leukocyte migration, was significantly decreased. KEGG enrichment results are as follows... Figure 20 As shown in D, in the ConA+iMSC-KO group of mice, NF-κB signaling, C-type lectin receptor signaling, IL-17 signaling, TNF-α signaling, and chemokine signaling pathways were significantly downregulated, while glycosphingolipid biosynthesis and mannosyl O-glycan biosynthesis were significantly upregulated. These results indicate that ConA+iMSC-KO can significantly reduce ConA-induced immune and inflammation-related signaling pathways and weaken leukocyte chemotaxis and migration, which is consistent with... Figure 18 The observed results were consistent. This tolerance to the immune response may be related to the loss of HLA-I molecules due to the knockout of the B2M gene.

[0132] In summary, the results indicate that iMSC-KO has a protective effect against ConA-induced liver injury. iMSC-KO significantly improved the survival rate of mice with liver injury, improved aspartate aminotransferase and alanine aminotransferase levels, significantly enhanced liver function in injured livers, reduced the level of the inflammatory factor TNF-α, decreased the area of ​​necrotic lesions, reduced inflammatory cell infiltration, reduced hepatocyte apoptosis, and decreased CD4+. + The level of T cells was also observed. Transcriptome sequencing analysis showed that iMSC-KO treatment significantly reduced signaling pathways related to lymphocyte activation, differentiation, and proliferation, while increasing the activity of metabolic signaling pathways. Furthermore, compared to the iMSC-WT treatment group, the iMSC-KO treatment group showed significantly downregulated signaling pathways related to immune response and leukocyte chemotaxis, indicating that iMSC-KO possesses higher immune tolerance. These results suggest that iMSC-KO can serve as an ideal cell source for treating liver injury.

[0133] 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 the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0134] 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. Use of induced pluripotent stem cell-derived mesenchymal stem cells in the preparation of a medicament for treating acute liver failure.

2. Use according to claim 1, characterized in that, The method for establishing the induced pluripotent stem cell-derived mesenchymal stem cells comprises the following steps: establishing an induced pluripotent stem cell line by reprogramming starting somatic cells, and obtaining a single clone of induced pluripotent stem cells by continuously picking single clones and subculturing; knocking out β2-microglobulin from the single clone of induced pluripotent stem cells to obtain a single clone of induced pluripotent stem cells with β2-microglobulin knocked out; after resuscitation and subculture, placing the single clone of induced pluripotent stem cells with β2-microglobulin knocked out in a directional differentiation medium for directional differentiation culture, and subculturing to the first generation of differentiated cells; subculturing the first generation of differentiated cells, and culturing to the fifth generation to obtain the mesenchymal stem cells.

3. Use according to claim 2, characterized in that, The starting somatic cells are umbilical cord blood mononuclear cells.

4. Use according to claim 2, characterized in that, The reprogramming kit used is CTS Cytotune Sendai reprogramming kit; and / or, the knocking out of β2-microglobulin uses CRISPR-Cas9 technology.

5. Use according to claim 2, characterized in that, The directional differentiation medium comprises the following components: Part 1: thaw StemFit basic 03-A basic medium and StemFit basic 03-B at 2-8℃ overnight, mix evenly in a biosafety cabinet, and distribute to 50mL centrifuge tubes; add CHIR99021 to 3μM, SB431542 to 10μM, and BMP4 to 50ng / mL; Part 2: thaw TBD medium additive at 2-8℃ overnight, mix the TBD basic medium and TBD additive evenly in a biosafety cabinet, and distribute to 50mL centrifuge tubes; add CHIR99021 to 3μM, SB431542 to 10μM, and L-Ascorbic acid to 100μM.

6. Use according to claim 1, characterized in that, The dosage form of the medicament is injection.

7. The use according to claim 1, characterized in that, The medicament takes the mesenchymal stem cells as the only active ingredient.

8. The use according to claim 1, characterized in that, The medicament is a medicament for improving aspartate aminotransferase and alanine aminotransferase levels.

9. The use according to claim 1, characterized in that, The medicament is a medicament for reducing the level of inflammatory factor TNF-α.

10. The use according to claim 1, characterized in that, The medicament is a medicament for reducing the level of apoptosis of liver cells.

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