Mesoderm differentiation specific human induced pluripotent stem cell and application thereof

Abstract

The invention relates to the technical field of induced pluripotent stem cells, in particular to a mesoderm differentiation specific human induced pluripotent stem cell and application thereof. The invention provides a human induced pluripotent stem cell BC-hiPSC-ME-20, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.46536. The invention further provides a preparation method of the human induced pluripotent stem cell BC-hiPSC-ME-20. The cell strain has multilineage differentiation potential and efficient mesoderm directional differentiation capacity, has stable pluripotency and excellent passage stability, has high safety, and has good application prospects in disease model construction, disease occurrence and development mechanism research, drug screening and cell therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of induced pluripotent stem cells, and particularly to a mesoderm differentiation-specific human induced pluripotent stem cell and its application. Background Art

[0002] Human induced pluripotent stem cells (hiPSCs), as a type of pluripotent stem cells, have the ability to differentiate into various cell types derived from the three germ layers (ectoderm, mesoderm, endoderm), bringing great potential to the fields of regenerative medicine, disease model construction, and drug screening. In recent years, the hiPSCs technology has received extensive attention in biomedical research and made remarkable progress.

[0003] The mesoderm plays a crucial role in embryonic development. It participates in the formation of various important organs and tissues, including the cardiovascular system, musculoskeletal system, urinary system, etc. In-depth study of the differentiation mechanism of mesoderm cells and the development of hiPSCs that can efficiently and specifically differentiate into mesoderm cells are of great significance for understanding the disease pathogenesis, developing new treatment methods, and exploring cell replacement therapies.

[0004] In addition, complex diseases involving mesoderm cells (such as cardiovascular diseases, muscular dystrophy diseases, musculoskeletal diseases, and kidney diseases, etc.) usually have a multi-factor pathogenic mechanism, involving the interaction of multiple cell types and signaling pathways. Existing disease models still face challenges in simulating these pathological processes. Therefore, the directed differentiation of hiPSCs into mesoderm cells helps to construct more accurate disease models, thus more realistically simulating disease progression and providing a more reliable research platform for mechanism research, drug screening, and cell therapy.

[0005] In summary, the development of an efficient, specific, and easy-to-operate mesoderm differentiation-specific hiPSCs is of great significance and application value for promoting the research and treatment of related diseases. Summary of the Invention

[0006] The present invention provides a mesoderm differentiation-specific human induced pluripotent stem cell and its application.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a human induced pluripotent stem cell BC-hiPSC-ME-20, which was deposited on May 21, 2025 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101), classified and named as Human Induced Pluripotent Stem Cells hiPSC, and the deposit number is CGMCC No. 46536.

[0009] In the present invention, a sample of peripheral blood mononuclear cells (PBMC) from a healthy human body was reprogrammed by introducing OCT4, SOX2, KLF4, and c-MYC, followed by clone formation, monoclonal screening, verification of pluripotency and differentiation characteristics, and evaluation of passage stability. Finally, a human induced pluripotent stem cell was obtained and named BC-hiPSC-ME-20. This cell line expresses a variety of stem cell markers, has the ability to differentiate into endoderm, mesoderm, and ectoderm cells, especially has a high differentiation efficiency in the directional differentiation into mesoderm cells, and can simply and efficiently prepare mesoderm cells; and has multi-lineage differentiation potential, can stably differentiate into mesenchymal stem cells (iMSC) and chondrocytes; and has high passage stability.

[0010] The above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 expresses at least one of the following stem cell markers: OCT4, NANOG, SOX2, TRA-1-81, SSEA-4, TRA-1-60.

[0011] The above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 can differentiate into cells of the three germ layers and has the ability of mesoderm-specific differentiation.

[0012] In a second aspect, the present invention provides progeny cells of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20.

[0013] Through passage experiments, it was verified that the human induced pluripotent stem cell BC-hiPSC-ME-20 has high passage stability, and its progeny cells have performance substantially equivalent to that of the human induced pluripotent stem cell BC-hiPSC-ME-20 cell line.

[0014] Preferably, the progeny cells are 1-50 generation progeny cells.

[0015] The present invention also provides a derivative cell of the human induced pluripotent stem cell BC-hiPSC-ME-20 or a derivative cell of a progeny cell thereof. The derivative cell is preferably a cell differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or a progeny cell thereof.

[0016] Cells differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or a progeny cell thereof include, but are not limited to, mesoderm, endoderm or ectoderm cells and derivative cells derived therefrom; for example, chondrocytes, induced mesenchymal stem cells (iMSCs), etc.

[0017] In a third aspect, the present invention provides the use of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 or the progeny cell, derivative cell or derivative cell of the progeny cell in the preparation of a pharmaceutical composition.

[0018] The active ingredient of the above-mentioned pharmaceutical composition comprises the human induced pluripotent stem cell BC-hiPSC-ME-20 or its culture, or comprises the progeny cell or its culture, or comprises a derivative cell of BC-hiPSC-ME-20 or a culture of a derivative cell thereof, or comprises a derivative cell of the progeny cell or a culture of a derivative cell thereof.

[0019] The above-mentioned pharmaceutical composition can be used for preventing and / or treating diseases of bone, cartilage, muscle, digestive system, urinary system, reproductive system, respiratory system, cardiovascular system or nervous system.

[0020] In a fourth aspect, the present invention provides the use of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 or the progeny cell, derivative cell or derivative cell of the progeny cell in the preparation of a cell therapy agent.

[0021] The active ingredient of the above-mentioned cell therapy agent comprises the human induced pluripotent stem cell BC-hiPSC-ME-20 or its culture, or comprises the progeny cell or its culture, or comprises a derivative cell of BC-hiPSC-ME-20 or a culture of a derivative cell thereof, or comprises a derivative cell of the progeny cell or a culture of a derivative cell thereof.

[0022] The above-mentioned cell therapy agent can be used for preventing and / or treating diseases of bone, cartilage, muscle, digestive system, urinary system, reproductive system, respiratory system, cardiovascular system or nervous system.

[0023] In the present invention, the cell therapy agent refers to cells isolated, cultured and prepared by special operations from the human body, and as a drug for preventive, therapeutic and diagnostic purposes, it can help restore cell or tissue function.

[0024] The above-mentioned pharmaceutical composition or cell therapy agent can be used for humans or animals.

[0025] In a fifth aspect, the present invention provides the use of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells, derivative cells or derivative cells of the progeny cells in the preparation of medical device products.

[0026] In a sixth aspect, the present invention provides the use of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells, derivative cells or derivative cells of the progeny cells in the preparation of disease cell models, organoids or stem cell transplantation animal models, or in the analysis of disease pathogenic mechanisms.

[0027] In the construction of the above-mentioned disease models, as an example, according to the disease type, the human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells of the present invention are directed to differentiate into specific cell types. For example, for cartilage diseases, they are differentiated into chondrocytes; for nervous system diseases, they are differentiated into neurons, glial cells, etc.; for cardiovascular diseases, they are differentiated into cardiomyocytes, endothelial cells, etc.; for hematological system diseases, they are differentiated into hematopoietic stem cells, etc. By using physical or chemical induction or gene editing, etc., the pathological characteristics of the disease are reproduced in vitro to construct a disease cell model. Similarly, the human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells can also be differentiated and cultured into organoids for disease model construction or drug screening.

[0028] In a seventh aspect, the present invention provides the use of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells, derivative cells or derivative cells of the progeny cells in drug screening or in the preparation of products for drug screening.

[0029] Using the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells to construct a disease cell model or an organoid can be used for the screening of disease prevention or treatment drugs.

[0030] In an eighth aspect, the present invention provides a cell therapeutic agent, which comprises any one or more of the following: (1) The above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20; (2) The culture of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20; (3) The progeny cells of the above-mentioned human induced pluripotent stem cell BC-hiPSC-ME-20, the derivative cells of BC-hiPSC-ME-20 or the derivative cells of the progeny cells; (4) Cultures of progeny cells of the human induced pluripotent stem cell BC-hiPSC-ME-20, cultures of derivative cells of BC-hiPSC-ME-20, or cultures of derivative cells of progeny cells.

[0031] The above-mentioned derivative cells are cells differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells, including but not limited to mesoderm, endoderm or ectoderm cells and their derived cells; for example, chondrocytes, mesenchymal stem cells (iMSC), etc.

[0032] In the present invention, the active ingredient of the cell therapeutic agent comprises any one or more of the above (1)-(4).

[0033] In a ninth aspect, the present invention provides a pharmaceutical composition, which comprises any one or more of the following: (1) The human induced pluripotent stem cell BC-hiPSC-ME-20; (2) Cultures of the human induced pluripotent stem cell BC-hiPSC-ME-20; (3) Progeny cells of the human induced pluripotent stem cell BC-hiPSC-ME-20, derivative cells of BC-hiPSC-ME-20 or derivative cells of progeny cells; (4) Cultures of progeny cells of the human induced pluripotent stem cell BC-hiPSC-ME-20, cultures of derivative cells of BC-hiPSC-ME-20, or cultures of derivative cells of progeny cells.

[0034] The above-mentioned derivative cells are cells differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells, including but not limited to mesoderm, endoderm or ectoderm cells and their derived cells; for example, chondrocytes, mesenchymal stem cells (iMSC), etc.

[0035] In the present invention, the active ingredient of the pharmaceutical composition comprises any one or more of the above (1)-(4).

[0036] In addition to the active ingredient, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may comprise emulsifying agents, suspending agents, preservatives, lubricants, wetting agents, etc. commonly used in formulations.

[0037] The pharmaceutical composition of the present invention can be administered by non-oral or oral routes. Among them, the non-oral administration can be carried out by injection (such as subcutaneous injection, intravenous injection, intramuscular injection, intraosseous injection, intra-articular injection, intraperitoneal injection), topical administration, endothelial administration, pulmonary administration, intranasal administration and rectal administration, etc.

[0038] In a tenth aspect, the present invention provides a medical device product, which includes any one or more of the following: (1) The human induced pluripotent stem cell BC-hiPSC-ME-20; (2) A culture of the human induced pluripotent stem cell BC-hiPSC-ME-20; (3) Progeny cells of the human induced pluripotent stem cell BC-hiPSC-ME-20, derivative cells of BC-hiPSC-ME-20, or derivative cells of progeny cells; (4) A culture of progeny cells of the human induced pluripotent stem cell BC-hiPSC-ME-20, a culture of derivative cells of BC-hiPSC-ME-20, or a culture of derivative cells of progeny cells.

[0039] The above-mentioned derivative cells are cells differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or its progeny cells, including but not limited to mesoderm, endoderm or ectoderm cells and their derived cells; for example, chondrocytes, induced mesenchymal stem cells (iMSC), etc.

[0040] The beneficial effects of the present invention at least include: The present invention provides a human induced pluripotent stem cell BC-hiPSC-ME-20 that can specifically differentiate into mesoderm cells. This cell line has good application prospects in disease model construction, research on the mechanisms of disease occurrence and development, drug screening, and cell therapy, providing new technical support for the development of translational medicine and regenerative medicine; the performance advantages of this cell line are mainly manifested in the following aspects: 1. High-efficiency and specific differentiation (1) Mesoderm-directed differentiation: BC-hiPSC-ME-20 exhibits excellent mesoderm differentiation ability (such as the score obtained according to the evaluation method of patent CN118910290B), significantly superior to the control cell lines (such as H1, ATCC-iPSC).

[0041] (2) Multilineage differentiation potential: It can stably differentiate into iMSC and chondrocytes, meeting the needs of regenerative medicine and the treatment of various diseases.

[0042] 2. Stable pluripotency (1) High expression of markers: The positive rates of OCT4 and NANOG are >99%, and the expression of surface markers (TRA-1-81, SSEA-4, TRA-1-60) is >93%, meeting international stem cell standards.

[0043] (2)Embryoid body formation ability: Successfully differentiated into cells of the three germ layers (such as PAX6 / Nestin+ in the ectoderm, Brachyury / NCAM+ in the mesoderm, and SOX17 / FOXA2+ in the endoderm).

[0044] (3)Teratoma formation ability: Teratomas can be formed after injecting BC-hiPSC-ME-20 cells into NOD-SCID mice, and typical three-germ layer structures can be seen in pathological analysis.

[0045] 3. Excellent passage stability (1)Long-term genetic stability: Maintained a normal karyotype after passage to P50.

[0046] (2)Functional consistency: The functions of iMSCs and chondrocytes differentiated from different passages are stable.

[0047] 4. Low risk and high safety Sendai virus (SeV) was detected negative for 3 consecutive passages, without virus residue, meeting the requirements for clinical-grade applications.

[0048] 5. Application advantages (1)Disease model construction: Provide highly realistic models for various mesoderm-related diseases.

[0049] (2)Drug screening platform: The high purity and stable functionality of differentiated cells improve the accuracy of drug testing.

[0050] (3)Clinical translation potential: Stable production processes and quality control standards (such as flow cytometry, qPCR, ICC) support GMP-grade production and clinical use. Description of the drawings

[0051] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0052] Figure 1 、 Figure 2 and Figure 3 are the cell morphology detection results of BC-hiPSC-ME-20 in Example 2 of the present invention; among them, Figure 1 、 Figure 2 and Figure 3 are the microscope observation results at 4×, 10×, and 20× in sequence.

[0053] Figure 4 and Figure 5Results of flow cytometry for detecting expression markers of BC-hiPSC-ME-20 in Example 2 of the present invention; among them, Figure 4 are the detection results of OCT4 and NANOG expression, Figure 5 are the detection results of SSEA-4, TRA-1-60, TRA-1-81, and CD45 expression; Figure 4 Among them, ISO-All represents the isotype control of the overall flow cytometry system, ISO-iPSC represents the isotype control of iPSC flow cytometry, ISO-Single represents the isotype control of iPSC single-cell flow cytometry, TEST-All represents the test sample of the overall flow cytometry system, TEST-iPSC represents the test sample of iPSC flow cytometry, TEST-Single represents the test sample of iPSC single-cell flow cytometry, and TEST-Live iPSC represents the test sample of iPSC live-cell flow cytometry.

[0054] Figure 6 、 Figure 7 and Figure 8 are the results of detecting the stem cell gene markers of BC-hiPSC-ME-20 by ICC method in Example 2 of the present invention; among them, Figure 6 is the detection result of OCT4, Figure 7 is the detection result of NANOG, Figure 8 is the detection result of SOX2; BF represents bright field.

[0055] Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 and Figure 13 are the detection results of embryoid body formation of BC-hiPSC-ME-20 in Example 2 of the present invention, among which, Figure 9 are the detection results of the ectoderm markers PAX6 and NESTIN (double staining), Figure 10 and Figure 11 are the detection results of the mesoderm markers NCAM and Brachyury, Figure 12 and Figure 13 are the detection results of the endoderm markers FOXA2 and SOX17.

[0056] Figure 14 are the results of evaluating the tridermal differentiation ability of BC-hiPSC-ME-20 in Example 3 of the present invention.

[0057] Figure 15 is the karyotype map of the P15 generation clone of BC-hiPSC-ME-20 cells in Example 4 of the present invention.

[0058] Figure 16 is the karyotype map of the P50 generation clone of BC-hiPSC-ME-20 cells in Example 4 of the present invention.

[0059] Figure 17 This is a graph showing the osteogenic, adipogenic and chondrogenic differentiation ability test results of MSCs in Example 4 of the present invention.

[0060] Figure 18 This is the morphology of the chondrocyte sphere in Example 4 of the present invention; wherein, 3D0d and 3D3d represent the 0th day and the 3rd day of preparing the chondrocyte sphere by the 3D induction method, respectively, and the same applies to other days.

[0061] Figure 19 This is the specific staining result of chondrocyte spheres in Example 4 of the present invention.

[0062] Figure 20 This is the detection result of the sGAG content of the key gene of the chondrocyte sphere in Example 4 of the present invention; wherein, 3D8d and 3D15d represent the 8th day and the 15th day of preparing the chondrocyte sphere by the 3D induction method, respectively, and the other days are similar. DETAILED DESCRIPTION

[0063] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] Example 1 Construction and screening of human induced pluripotent stem cells In this example, Sendai virus was used to reprogram PBMC to construct human induced pluripotent stem cells, and the BC-hiPSC-ME-20 cell line was obtained by screening. The specific process is as follows: 1. Isolation and cryopreservation of PBMC (1) Blood collection and PBMC isolation: Healthy donors were screened, peripheral blood was collected, PBMCs were separated by Ficoll density gradient centrifugation (900 × g, 20 min), and washed with DPBS containing 1% human albumin.

[0065] (2) Counting and freezing: After PBMCs are counted, they are stored in a certain size (e.g. 8×10 7 The cells were divided into 200 μl and 100 μl (viable cells / mL) and packaged with CryoStor CS10 freezing medium and stored in a vapor phase liquid nitrogen tank.

[0066] 2. PBMC recovery and pre-culture (1) Recovery: Frozen PBMCs were rapidly thawed in a 37°C water bath, resuspended in PBMC complete medium (containing SCF, IL-3, IL-6 and other cytokines), and inoculated into 12-well plates (2 × 106 cells / mL).

[0067] (2) Pre-stimulation: Continuously culture for 3 days (Day -4 to Day -1), and change the culture medium daily to activate cell proliferation.

[0068] 3. Sendai virus transduction (Day 0) (1) Virus preparation: Thaw the Sendai virus kit (carrying OCT4 / SOX2 / KLF4 / c-MYC), and calculate the dosage according to MOI = 5.

[0069] (2) Centrifugal infection: Mix PBMC with the virus and centrifuge (1000×g, 30 min), then inoculate it into a 12-well plate and culture at 37°C for 18 - 26 hours.

[0070] 4. Reprogramming initiation (Day 1 - 7) (1) Virus removal (Day 1): Change the complete culture medium of PBMC to remove free virus.

[0071] (2) Cell passage (Day 3): Inoculate the cells at a gradient density (1×10 4 to 5×10 4 cells / mL) into a 6-well plate coated with Vitronectin.

[0072] (3) Culture medium conversion (Day 5 - 7): Day 5: Change to PBMC factor-free culture medium.

[0073] Day 7: Switch to TeSR™-AOF complete culture medium to induce pluripotency.

[0074] 5. Human induced pluripotent stem cell colony formation and selection (Day 8 - 20) (1) Update the culture medium daily: Aspirate the old culture medium and add fresh TeSR™-AOF (2 mL / well).

[0075] (2) Colony selection (Day 17 - 20): Manually pick typical iPSC colonies (clear edges, high nuclear-cytoplasmic ratio) and inoculate them into a 12-well plate (1 colony / well, P0 generation), and the culture medium contains 10 μM Y-27632 (anti-apoptosis).

[0076] 6. iPSC clone expansion and cryopreservation (1) Passage (P01 generation): When the P0 generation cells reach 10 - 20% confluence, dissociate them with TrypLE and inoculate them into a 6-well plate for expansion.

[0077] (2)Cryopreservation: When the P01 cells are fused to 70 - 90%, they are cryopreserved with CryoStor CS10 (such as 1.85×10 6 cells / vial).

[0078] 7. Monoclonal screening: Four clones with the best morphology and cryopreservation quantity are selected. 48 sub - clones derived from single cells are picked by low - density inoculation. Through multiple consecutive manual selections of single - cell clones, multiple clones with negative Sendai virus detection results for three consecutive generations are obtained.

[0079] 8. Verification of pluripotency and differentiation: The iPSC markers of different clones (OCT4 / NANOG expression > 85%) and the differentiation abilities into mesenchymal stem cells (iMSC) and chondrocytes are detected, and 7 candidate sub - clones are screened out.

[0080] 9. STR testing: The STR results show that (Table 1) there are no four alleles at each locus, and no human cell cross - contamination is found in the cell line. The DNA typing is 100% matched with PBMC cells, and they are homologous cells.

[0081] Table 1

[0082] 10. Evaluation of passage stability: The 7 sub - clones are passaged long - term (to P50), and the karyotype, gene mutations, and differentiation function stability are monitored. Finally, the sub - clone that maintains a normal karyotype, has no pathogenic mutations, and has continuous and stable pluripotency and directed differentiation ability during passage is selected as the seed cell for research, and it is named BC - hiPSC - ME - 20.

[0083] The human induced pluripotent stem cell BC - hiPSC - ME - 20 was deposited on May 21, 2025 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101). It is classified and named as Human Induced Pluripotent Stem Cells hiPSC, and the deposit number is CGMCC No. 46536.

[0084] Example 2 Detection of the basic properties of human induced pluripotent stem cell BC - hiPSC - ME - 20 The basic properties of the human induced pluripotent stem cell BC - hiPSC - ME - 20 obtained in Example 1, such as content, growth characteristics, cell identification, biological characteristics, and impurities, are detected. The specific detection methods and results are shown in Table 2.

[0085] The detection methods shown in Table 2 are all conventional methods for human induced pluripotent stem cell detection. Among them, the method for embryoid body formation detection is as follows: For BC-hiPSC-ME-20 with a growth confluence of 80-90% in a 6-well plate, after cross-cutting using a mechanical method, it was cultured in suspension in EB complete medium for 7 days. An appropriate amount of EB spheres were inoculated into a 24-well plate with EB complete medium and cultured for 1 day for adhesion. The adhered cells were fixed, permeabilized, blocked, incubated with the corresponding primary antibody, incubated with the corresponding secondary antibody, stained with nuclear DAPI, and photographed under fluorescence.

[0086] Table 2 Detection results of BC-hiPSC-ME-20

[0087] Among them, the detection results of cell morphology are as shown in Figure 1 、 Figure 2 and Figure 3 ; the results of flow cytometry for detecting expression markers are as shown in Figure 4 and Figure 5 ; the results of ICC method for detecting stemness genes are as shown in Figure 6 、 Figure 7 and Figure 8 ; the detection results of embryoid body formation are as shown in Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 and Figure 13 .

[0088] Example 3 Detection of mesoderm-specific differentiation ability of human induced pluripotent stem cell BC-hiPSC-ME-20 The mesoderm-specific differentiation ability of BC-hiPSC-ME-20 was detected using the method for evaluating the pluripotency of pluripotent stem cells (see Example 3 of Patent CN118910290B). H1 (hESC human embryonic stem cells, donated by Tsinghua University, publicly disclosed in the following literature: Hu BY, Weick JP, Yu J, Ma LX, Zhang XQ, Thomson JA, Zhang SC. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc Natl Acad Sci USA. 2010 Mar 2;107(9):4335-40. doi:10.1073 / pnas.0910012107. Epub 2010 Feb 16. PMID:20160098; PMCID:PMC2840097) and ATCC-iPSC (human induced pluripotent stem cell HYS0103, purchased from ATCC) were used as controls.

[0089] The results showed ( Figure 14 ), BC-hiPSC-ME-20 had the specific differentiation ability into mesoderm (such as iMSC and chondrocytes), which was significantly better than the control cell lines (H1, ATCC-iPSC).

[0090] Example 4 Verification of the Differentiation Ability and Passage Stability of BC-hiPSC-ME-20 1. Results of Karyotype Detection The karyotypes of BC-hiPSC-ME-20 cells at different passages were detected using the chromosome G-banding method (select 50 metaphase cells for photomicrography, perform karyotype analysis, and roughly count 500 metaphase cells to check the incidence of polyploidy). The detection results are shown in Table 3 and all meet the requirements.

[0091] Table 3

[0092] The karyotype map of the P15 generation clone of BC-hiPSC-ME-20 cells is as Figure 15 shown.

[0093] The karyotype map of the P50 generation clone of BC-hiPSC-ME-20 cells is as Figure 16 shown.

[0094] 2. Directed Inductive Differentiation of BC-hiPSC-ME-20 into iMSC Induce the differentiation of BC-hiPSC-ME-20 into iMSC, and the specific method is as follows: Resuspend BC-hiPSC-ME-20 cells with Essential-8™ complete medium (Thermo Fisher, A1517001), and then inoculate them into a vitronectin-coated cell culture dish for cell amplification. When the confluence of BC-hiPSC-ME-20 cells reaches 90%, add CTS™ TrypLE™ Select digestive enzyme (Thermo Fisher, A1285901) to collect BC-hiPSC-ME-20 cells and prepare a single-cell suspension, and then inoculate BC-hiPSC-ME-20 cells into a six-well plate coated with type IV collagen (Sigma, C6745). The medium is changed to a differentiation medium: IMDM (Thermo Fisher, 12440053) containing Ham’s F-12 Nutrient Mix (Thermo Fisher, 11765054), BMP4 (Peprotech, 120-05), and Activin A (Peprotech, AF-120-14E-1MG). Subsequently, transfer the cells to a mesenchymal colony-forming semi-solid medium containing Stem-Span TM serum-free expansion medium (Stemcell, 09650), ES-Cult TM M3120 (Stemcell, 03120), ESFM human endothelial serum-free medium (Thermo Fisher, 11111044), and FGF-2 (Stemimmune, HST-F2-1000), and continue to culture until spherical mesenchymal vascular cell colonies are formed. The cell colonies are collected through a 100 μM cell sieve, and then resuspended with a mesenchymal stem cell serum-free medium containing StemLine II (Sigma, S0192), human ESFM, and FGF-2, and inoculated into a fibronectin / collagen-coated culture flask. The finally collected iMSC are cryopreserved in a liquid nitrogen tank.

[0095] Use flow cytometry to detect the cell surface markers of iMSC differentiated from different passages (P13, P17, P21) of BC-hiPSC-ME-20. The detection results show (Table 4): The positive indicators of MSC, CD90, CD73, CD105, and CD44, are all greater than 95%; the negative indicators, CD11B, CD19, CD31, CD34, CD45, and HLADR, are all less than 2%, meeting the MSC property requirements.

[0096] Table 4

[0097] The MSC cells differentiated from BC-hiPSC-ME-20 at different passages (P13, P17, P21) were subjected to trilineage (adipogenic, osteogenic, chondrogenic) differentiation assays. The results showed that ( Figure 17 ), the MSC cells differentiated from BC-hiPSC-ME-20 at different passages (P13, P17, P21) all had the ability of adipogenic, osteogenic, and chondrogenic differentiation, meeting the property requirements of MSCs.

[0098] 3. Directed induction of BC-hiPSC-ME-20 into chondrocytes BC-hiPSC-ME-20 was directed to differentiate into chondrocyte spheres according to the method in Example 1 of Patent CN118109397B.

[0099] The morphology of the chondrocyte spheres was as Figure 18 shown. The results of specific staining were as Figure 19 shown. The detection results of the change in the content of the key gene sGAG in the chondrocyte spheres (sGAG content normalized to wet weight) were as Figure 20 shown. The results showed that the sGAG content in the chondrocyte spheres showed a gradually increasing trend with the prolongation of the induction time and remained stable at 3D46d.

[0100] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Human induced pluripotent stem cell BC-hiPSC-ME-20, characterized in that, It is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 46536.

2. The human induced pluripotent stem cell BC-hiPSC-ME-20 according to claim 1, characterized in that, The human induced pluripotent stem cell BC-hiPSC-ME-20 expresses at least one selected from the following stem cell markers: OCT4, NANOG, SOX2, TRA-1-81, SSEA-4, TRA-1-60.

3. The human induced pluripotent stem cell BC-hiPSC-ME-20 according to claim 1 or 2, characterized in that, The human induced pluripotent stem cell BC-hiPSC-ME-20 can differentiate into cells of the three germ layers and has the ability of mesoderm-specific differentiation.

4. Progeny cells, derivative cells or derivative cells of progeny cells of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3.

5. Use of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3 or the progeny cells, derivative cells or derivative cells of progeny cells according to claim 4 in the preparation of a pharmaceutical composition or a cell therapeutic agent.

6. Use of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3 or the progeny cells, derivative cells or derivative cells of progeny cells according to claim 4 in the preparation of medical device products.

7. Use of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3 or the progeny cells, derivative cells or derivative cells of progeny cells according to claim 4 in the preparation of a disease cell model, an organoid or a stem cell transplantation animal model, or in the analysis of the pathogenic mechanism of a disease.

8. Use of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3 or the progeny cells, derivative cells or derivative cells of progeny cells according to claim 4 in drug screening or the preparation of products for drug screening.

9. A pharmaceutical composition or cell therapy agent, characterized in that, The pharmaceutical composition or the cell therapeutic agent contains any one or more of the following: (1) The human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3; (2) A culture of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3; (3) The progeny cells, derivative cells or derivative cells of progeny cells according to claim 4; (4) A culture of the progeny cells, derivative cells or derivative cells of progeny cells according to claim 4.

10. A medical device product, characterized in that, The medical device products contain any one or more of the following: (1) The human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3; (2) A culture of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3; (3) The progeny cells, derivative cells or derivative cells of progeny cells according to claim 4; (4) A culture of the progeny cells, derivative cells or derivative cells of progeny cells according to claim 4.

Images