Mesoderm differentiation specific human induced pluripotent stem cells and uses thereof

Abstract

The present application relates to the technical field of induced pluripotent stem cell, and particularly relates to mesoderm differentiation specific human induced pluripotent stem cell and application thereof. The present application provides human induced pluripotent stem cell BC-hiPSC-ME-20, which is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.46536. The cell strain has multi-lineage differentiation potential and high-efficiency mesoderm directional differentiation capacity, has stable pluripotency and excellent passage stability, and has higher safety, and has a good application prospect in disease model construction, disease occurrence and development mechanism research, drug screening and cell therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of induced pluripotent stem cell technology, in particular to a mesoderm differentiation specific human induced pluripotent stem cell and its application. BACKGROUND

[0002] Human induced pluripotent stem cells (hiPSCs) are a type of stem cell with pluripotency, which 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, hiPSCs technology has received extensive attention in biomedical research and has made significant progress.

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

[0004] In addition, complex diseases related to mesoderm cells (such as cardiovascular diseases, muscle atrophy diseases, musculoskeletal diseases, and kidney diseases) often have multifactorial pathogenic mechanisms, involving the interaction of multiple cell types and signaling pathways. Existing disease models still face challenges in simulating these pathological processes. Therefore, using hiPSCs to differentiate into mesoderm cells can help 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, developing 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

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

[0007] Specifically, the present application provides the technical solutions described below.

[0008] In a first aspect, the present application provides a human induced pluripotent stem cell BC-hiPSC-ME-20, which was deposited on May 21, 2025 at the China General Microbiological Culture Collection Center (CGMCC, located at No. 1, Yihuangyuan, Beichenxi Road, Beijing, China, and managed by the Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China) and classified as Human Induced Pluripotent Stem Cells hiPSC with a deposit number of CGMCC No. 46536.

[0009] The present application reprograms a sample of peripheral blood mononuclear cells (PBMC) from a healthy human body, introduces OCT4, SOX2, KLF4 and c-MYC, and performs cloning formation and monoclonal screening, as well as pluripotency and differentiation property verification and passage stability evaluation, to finally obtain a human induced pluripotent stem cell, which is named BC-hiPSC-ME-20. The cell strain expresses various stem cell markers, has the ability to differentiate into endodermal, mesodermal and ectodermal cells, especially has a high differentiation efficiency in the direction of mesodermal cells, and can be simply and efficiently prepared into mesodermal cells; and has a multi-lineage differentiation potential, can be stably differentiated into mesenchymal stem cells (iMSC) and chondrocytes; and has a high passage stability.

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

[0011] The human induced pluripotent stem cell BC-hiPSC-ME-20 described above can differentiate into three germ layer cells and has mesodermal specific differentiation ability.

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

[0013] The present application verifies through passage experiments that the human induced pluripotent stem cell BC-hiPSC-ME-20 has a high passage stability, and the progeny cells thereof have substantially equivalent performance to the cell strain of the human induced pluripotent stem cell BC-hiPSC-ME-20.

[0014] Preferably, the progeny cell is a 1-50th progeny cell.

[0015] The present application also provides a derivative cell of a derivative cell of the human induced pluripotent stem cell BC-hiPSC-ME-20 or 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] The cell differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or a progeny cell thereof includes, but is not limited to, mesoderm, endoderm or ectoderm cells and derivative cells derived therefrom; for example, chondrocytes, mesenchymal stem cells (iMSC) and the like.

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

[0018] The effective component of the above-mentioned pharmaceutical composition comprises the human induced pluripotent stem cell BC-hiPSC-ME-20 or a culture thereof, or comprises the progeny cell or a culture thereof, or comprises a derivative cell of BC-hiPSC-ME-20 or a culture of the derivative cell thereof, or comprises a derivative cell of the progeny cell or a culture of the 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 application provides use of the human induced pluripotent stem cell BC-hiPSC-ME-20 or the progeny cell, derivative cell or derivative cell of the progeny cell described above in the preparation of a cell therapeutic agent.

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

[0022] The above-mentioned cell therapeutic 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 application, the cell therapeutic agent refers to a cell isolated, cultured and prepared by special operation from the human body, which can help restore cell or tissue function as a drug for prevention, treatment and diagnosis purposes.

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

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

[0026] In a sixth aspect, the present application provides use of the human induced pluripotent stem cell BC-hiPSC-ME-20 or the progeny cells, derivative cells or derivative cells of the progeny cells described above in the preparation of a disease cell model, organoid or stem cell transplantation animal model, or in the analysis of disease pathogenesis.

[0027] In the construction of the disease model described above, as an example, according to the type of disease, the human induced pluripotent stem cell BC-hiPSC-ME-20 or the progeny cells described in the present application are differentiated into specific cell types. For example, for cartilage diseases, chondrocytes are differentiated; for nervous system diseases, neurons, glial cells, etc. are differentiated; for cardiovascular diseases, cardiomyocytes, endothelial cells, etc. are differentiated; for blood system diseases, hematopoietic stem cells, etc. are differentiated. 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 the progeny cells can also be differentiated and cultured into organoids for disease model construction or drug screening.

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

[0029] The disease cell model or organoid constructed using the human induced pluripotent stem cell BC-hiPSC-ME-20 or the progeny cells described above can be used for the screening of disease prevention or treatment drugs.

[0030] In an eighth aspect, the present application provides a cell therapeutic agent, which comprises any one or more of the following:

[0031] (1) the human induced pluripotent stem cell BC-hiPSC-ME-20;

[0032] (2) a culture of the human induced pluripotent stem cell BC-hiPSC-ME-20;

[0033] (3) a progeny cell of the human induced pluripotent stem cell BC-hiPSC-ME-20, a derivative cell of BC-hiPSC-ME-20 or a derivative cell of the progeny cell;

[0034] (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.

[0035] The derivative cells described above are cells differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or progeny cells thereof, including but not limited to mesoderm, endoderm or ectoderm cells and derivative cells derived therefrom; for example, chondrocytes, mesenchymal stem cells (iMSCs), and the like.

[0036] In the present application, the effective component of the cell therapeutic agent contains any one or more of the above (1) to (4).

[0037] In the ninth aspect, the present application provides a pharmaceutical composition containing any one or more of the following:

[0038] (1) the human induced pluripotent stem cell BC-hiPSC-ME-20;

[0039] (2) a culture of the human induced pluripotent stem cell BC-hiPSC-ME-20;

[0040] (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;

[0041] (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.

[0042] The derivative cells described above are cells differentiated from the human induced pluripotent stem cell BC-hiPSC-ME-20 or progeny cells thereof, including but not limited to mesoderm, endoderm or ectoderm cells and derivative cells derived therefrom; for example, chondrocytes, mesenchymal stem cells (iMSCs), and the like.

[0043] In the present application, the effective component of the pharmaceutical composition contains any one or more of the above (1) to (4).

[0044] In addition to the effective component, the pharmaceutical composition can further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can contain emulsifiers, suspending agents, preservatives, lubricants, wetting agents, and the like, which are commonly used in formulation.

[0045] The pharmaceutical composition of the present application can be administered in a non-oral or oral manner. Among them, the non-oral administration can be administered by injection (such as subcutaneous injection, intravenous injection, intramuscular injection, intraosseous injection, intraarticular injection, intraperitoneal injection), local administration, endothelial administration, pulmonary administration, intranasal administration and rectal administration, etc.

[0046] In a tenth aspect, the present application provides a medical device product, which comprises any one or more of the following:

[0047] (1) the human induced pluripotent stem cell BC-hiPSC-ME-20;

[0048] (2) the culture of the human induced pluripotent stem cell BC-hiPSC-ME-20;

[0049] (3) the progeny cells of the 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;

[0050] (4) the culture of the progeny cells of the human induced pluripotent stem cell BC-hiPSC-ME-20, the culture of the derivative cells of BC-hiPSC-ME-20 or the culture of the derivative cells of the progeny cells.

[0051] The derivative cells mentioned above 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 derivative cells derived therefrom; for example, chondrocytes, mesenchymal stem cells (iMSC) and the like.

[0052] The beneficial effects of the present application at least include: the present application provides a human induced pluripotent stem cell BC-hiPSC-ME-20 which can be specifically differentiated into mesoderm cells, and the cell strain has good application prospect in disease model construction, disease occurrence and development mechanism research, drug screening and cell therapy, and provides new technical support for the development of translational medicine and regenerative medicine; the performance advantages of the cell strain mainly manifest in the following aspects:

[0053] 1. High-efficiency specific differentiation

[0054] (1) Mesoderm directional differentiation: BC-hiPSC-ME-20 exhibits excellent mesoderm differentiation ability (for example, the score obtained according to the evaluation method of patent CN118910290B), which is significantly better than the control cell line (such as H1, ATCC-iPSC).

[0055] (2) Multilineage differentiation potential: can be stably differentiated into iMSC and chondrocytes, meeting the needs of regenerative medicine and treatment of various diseases.

[0056] 2. Stable pluripotency

[0057] (1) High expression of markers: OCT4, NANOG positive rate > 99%, surface markers (TRA-1-81, SSEA-4, TRA-1-60) expression > 93%, in line with international stem cell standards.

[0058] (2) Embryoid body formation ability: successfully differentiated into three germ layer cells (such as ectoderm PAX6 / Nestin+, mesoderm Brachyury / NCAM+, endoderm SOX17 / FOXA2+).

[0059] (3) Teratoma formation ability: NOD-SCID mice injected with BC-hiPSC-ME-20 cells can form teratomas, and pathological analysis can see typical three germ layer structure.

[0060] 3. Excellent passaging stability

[0061] (1) Long-term genetic stability: normal karyotype maintained after P50 passage.

[0062] (2) Functional consistency: iMSC and chondrocyte functions of different passages are stable.

[0063] 4. Low risk and high safety

[0064] Sendai virus (SeV) was negative after 3 consecutive passages, with no virus residue, meeting the requirements of clinical level application.

[0065] 5. Application advantages

[0066] (1) Disease model construction: provides high simulation models for various mesoderm-related diseases.

[0067] (2) Drug screening platform: high purity and stable functionality of differentiated cells improve drug testing accuracy.

[0068] (3) Clinical transformation potential: stable production process and quality control standards (such as flow cytometry, qPCR, ICC) support GMP-level production and clinical use. BRIEF DESCRIPTION OF DRAWINGS

[0069] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0070] Figure 1 、 Figure 2 andFigure 3 The cell morphology detection results of BC-hiPSC-ME-20 in Example 2 of the present application; wherein, Figure 1 , Figure 2 and Figure 3 are the microscope observation results of 4x, 10x, 20x, respectively.

[0071] Figure 4 and Figure 5 are the flow cytometry detection results of BC-hiPSC-ME-20 expressing markers in Example 2 of the present application; wherein, Figure 4 is the detection result of OCT4 and NANOG expression, Figure 5 is the detection result of SSEA-4, TRA-1-60, TRA-1-81 and CD45 expression; Figure 4 In the table, ISO-All represents the whole flow system isotype control, ISO-iPSC represents the iPSC flow isotype control, ISO-Single represents the iPSC single cell flow isotype control, TEST-All represents the whole flow system test sample, TEST-iPSC represents the iPSC flow test sample, TEST-Single represents the iPSC single cell flow test sample, and TEST-Live iPSC represents the iPSC live cell flow test sample.

[0072] Figure 6 , Figure 7 and Figure 8 are the results of ICC method for detecting the stemness gene markers of BC-hiPSC-ME-20 in Example 2 of the present application; wherein, Figure 6 is the OCT4 detection result, Figure 7 is the NANOG detection result, Figure 8 is the SOX2 detection result; BF represents the bright field.

[0073] Figure 9 , Figure 10 , Figure 11 , Figure 12 and Figure 13 are the results of the embryoid body formation detection of BC-hiPSC-ME-20 in Example 2 of the present application, wherein, Figure 9 is the detection result of ectoderm marker PAX6 and NESTIN (double staining), Figure 10 and Figure 11 are the detection results of mesoderm markers NCAM and Brachyury, Figure 12 and Figure 13 are the detection results of endoderm markers FOXA2 and SOX17.

[0074] Figure 14The results show the trigerm layer differentiation capacity assessment of BC-hiPSC-ME-20 in Example 3 of this invention.

[0075] Figure 15 This is a karyotype diagram of the chromosomes of the P15 generation clone of BC-hiPSC-ME-20 cells in Example 4 of the present invention.

[0076] Figure 16 This is a karyotype diagram of the chromosomes of the P50 generation clone of BC-hiPSC-ME-20 cells in Example 4 of the present invention.

[0077] Figure 17 This is a graph showing the detection results of osteogenic, adipogenic, and chondrogenic differentiation capacity of MSCs in Example 4 of the present invention.

[0078] Figure 18 The image shows the morphology of chondrocyte spheres in Example 4 of this invention; where 3D0d and 3D3d represent day 0 and day 3 of chondrocyte sphere preparation by the 3D induction method, respectively, and other days follow the same pattern.

[0079] Figure 19 This is the specific staining result of chondrocyte spheres in Example 4 of the present invention.

[0080] Figure 20 The results show the detection results of the sGAG content of the key gene in chondrocyte spheroids in Example 4 of this invention; where 3D8d and 3D15d represent the 8th and 15th days of chondrocyte spheroids prepared by the 3D induction method, respectively, and other days are deduced accordingly. Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0082] Example 1: Construction and screening of human induced pluripotent stem cells

[0083] In this embodiment, human induced pluripotent stem cells were constructed by reprogramming PBMCs with Sendai virus, and the BC-hiPSC-ME-20 cell line was obtained through screening. The specific process is as follows:

[0084] 1. Isolation and cryopreservation of PBMCs

[0085] (1) Blood collection and PBMC isolation: healthy donors were screened and peripheral blood was collected. PBMC were isolated by Ficoll density gradient centrifugation (900 x g, 20 min) and washed with DPBS containing 1% human albumin.

[0086] (2) Counting and cryopreservation: after counting, PBMC were aliquoted with CryoStor CS10 cryopreservation solution at a certain specification (e.g. 8 x 10 7 cells / mL) and stored in a gas-phase liquid nitrogen tank.

[0087] 2. PBMC recovery and pre-culture

[0088] (1) Recovery: cryopreserved PBMC were quickly thawed at 37°C water bath, resuspended with PBMC complete medium (containing SCF, IL-3, IL-6, etc. cytokines) and seeded into 12-well plates (2 x 10 6 cells / mL).

[0089] (2) Pre-stimulation: continuously cultured for 3 days (Day -4 to Day -1), and the medium was changed daily to activate cell proliferation.

[0090] 3. Sendai virus transduction (Day 0)

[0091] (1) Virus preparation: thaw Sendai virus kit (carrying OCT4 / SOX2 / KLF4 / c-MYC), calculate the dosage according to MOI = 5.

[0092] (2) Centrifugal infection: after mixing PBMC with virus, centrifugation (1000 x g, 30 min), seeded into 12-well plates, and cultured at 37°C for 18-26 hours.

[0093] 4. Reprogramming initiation (Day 1-7)

[0094] (1) Virus removal (Day 1): change PBMC complete medium to remove free virus.

[0095] (2) Cell passage (Day 3): cells were seeded into Vitronectin-coated 6-well plates at a gradient density (1 x 10 4 to 5 x 10 4 cells / mL).

[0096] (3) Medium conversion (Day 5-7):

[0097] Day 5: changed to PBMC factor-free medium.

[0098] Day 7: switched to TeSR™-AOF complete medium to induce pluripotency.

[0099] 5. Human induced pluripotent stem cell clone formation and picking (Day 8-20)

[0100] (1) Daily medium refreshment: discard old medium, add fresh TeSR™-AOF (2 mL / well).

[0101] (2) Clone picking (Day 17-20): manually pick morphologically typical iPSC colonies (clear edge, high nuclear-cytoplasmic ratio) and seed into 12-well plates (1 colony / well, P0 passage) with medium containing 10 μΜ Y-27632 (anti-apoptotic).

[0102] 6. iPSC clone expansion and cryopreservation

[0103] (1) Passage (P01 passage): when P0 cells reach 10-20% confluence, dissociate with TrypLE and seed into 6-well plates for expansion.

[0104] (2) Cryopreservation: when P01 cells reach 70-90% confluence, cryopreserve with CryoStor CS10 (e.g. 1.85 x 10 6 cells / stick).

[0105] 7. Single clone screening: select 4 clones with the best morphology and cryopreservation amount, pick 48 sub-clones from single cells by low-density seeding, and obtain multiple clones with negative results for continuous three passages of Sendai virus detection by multiple continuous manual picking of single cell clones.

[0106] 8. Pluripotency and differentiation verification: detect the iPSC markers (OCT4 / NANOG expression > 85%) and differentiation ability of mesenchymal stem cells (iMSC) and chondrocytes of different clones, and select 7 candidate sub-clones.

[0107] 9. STR test: the STR results (Table 1) show that there are no four alleles at each locus, and no human cell cross-contamination is found in the cell line. The DNA typing matches the PBMC cells 100%, which are homologous cells.

[0108] Table 1

[0109]

[0110] 10. Passage stability evaluation: long-term passage (to P50) is performed on 7 sub-clones, and the karyotype, gene mutation, and differentiation function stability are monitored, and finally the sub-clones that maintain normal karyotype, no pathogenic mutation, and continuous stability of pluripotency and directional differentiation ability during passage are selected as research seed cells, which are named BC-hiPSC-ME-20.

[0111] The human induced pluripotent stem cells BC-hiPSC-ME-20 were preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 21, 2025, and were classified and named as human induced pluripotent stem cells Human Induced Pluripotent Stem Cells hiPSC, with the preservation number of CGMCC No. 46536.

[0112] Example 2. Basic performance detection of human induced pluripotent stem cells BC-hiPSC-ME-20

[0113] The basic performance of the human induced pluripotent stem cells BC-hiPSC-ME-20 obtained in Example 1, such as content, growth characteristics, cell identification, biological characteristics, and impurities, was detected, and the specific detection methods and detection results are shown in Table 2.

[0114] The detection methods shown in Table 2 are all conventional methods for detecting human induced pluripotent stem cells, wherein the method for detecting embryoid body formation is as follows:

[0115] The BC-hiPSC-ME-20 in the 6-well plate with a growth confluence of 80-90% was cross-cut using a mechanical method, then suspended in EB complete medium for 7 days, and then a suitable amount of EB balls were inoculated in a 24-well plate using EB complete medium for 1 day for adhesion. The cells after adhesion were fixed, permeabilized, blocked, incubated with the corresponding primary antibody, incubated with the corresponding secondary antibody, and stained with DAPI, and then photographed under fluorescence.

[0116] Table 2. Detection results of BC-hiPSC-ME-20

[0117]

[0118] The cell morphology detection results are shown in Figure 1 , Figure 2 and Figure 3 , the flow cytometry detection results of the expression markers are shown in Figure 4 and Figure 5 , the ICC method detection results of the stemness genes are shown in Figure 6 , Figure 7 and Figure 8 , and the embryoid body formation detection results are shown in Figure 9 , Figure 10 , Figure 11 , Figure 12 and Figure 13 .

[0119] Example 3 Detection of mesoderm-specific differentiation ability of human induced pluripotent stem cells BC-hiPSC-ME-20

[0120] The mesoderm-specific differentiation ability of BC-hiPSC-ME-20 was detected by the method for evaluating pluripotency of pluripotent stem cells (see Example 3 of patent CN118910290B), with H1 (hESC human embryonic stem cells, from Tsinghua University (donated), 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 U S A. 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 cells HYS0103, purchased from ATCC) as controls.

[0121] The results show that (Table 4) Figure 14 ), BC-hiPSC-ME-20 has specific mesoderm (such as iMSC and chondrocyte) differentiation ability, which is significantly better than the control cell lines (H1, ATCC-iPSC).

[0122] Example 4 Verification of differentiation ability and passage stability of BC-hiPSC-ME-20

[0123] 1. Karyotype detection results

[0124] Karyotype detection was performed on BC-hiPSC-ME-20 cells of different passages by chromosome G banding method (50 metaphase cells were selected for microphotography, karyotype analysis was made, and 500 metaphase cells were roughly counted to check the incidence of polyploidy). The detection results are shown in Table 3, which all meet the requirements.

[0125] Table 3

[0126]

[0127] The karyotype of the P15 generation clone of BC-hiPSC-ME-20 cells is shown in Figure 15 .

[0128] The karyotype diagram of the chromosomes of the P50 generation clones in BC-hiPSC-ME-20 cells is shown below. Figure 16 As shown.

[0129] 2. Directed differentiation of BC-hiPSC-ME-20 into iMSCs

[0130] The BC-hiPSC-ME-20 was induced to differentiate into iMSCs using the following method:

[0131] BC-hiPSC-ME-20 cells were resuspended in Essential-8™ complete medium (Thermo Fisher, A1517001) and then seeded into vitronectin-coated cell culture dishes for cell expansion. When the confluence of BC-hiPSC-ME-20 cells reached 90%, CTS™ TrypLE™ Select digestive enzyme (Thermo Fisher, A1285901) was added to collect BC-hiPSC-ME-20 cells and prepare a single-cell suspension. Then, BC-hiPSC-ME-20 cells were seeded into six-well plates coated with type IV collagen (Sigma, C6745), and the culture medium was replaced with 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). The cells were then transferred to Stem-Span... TM Serum-free amplification medium (Stemcell, 09650), ES-Cult TM Mesenchymal stem cell (iMSCs) were cultured in a semi-solid culture medium containing M3120 (Stemcell, 03120), serum-free ESFM human endothelial cell medium (Thermo Fisher, 11111044), and FGF-2 (Stemimmune, HST-F2-1000) until spherical mesenchymal vascular cell colonies formed. Cell colonies were collected through a 100 μM cell sieve, resuspended in serum-free mesenchymal stem cell medium containing StemLine II (Sigma, S0192), human ESFM, and FGF-2, and seeded into fibronectin / collagen-coated culture flasks. The collected iMSCs were then cryopreserved in liquid nitrogen.

[0132] The iMSC obtained by different passages (P13, P17, P21) of BC-hiPSC-ME-20 differentiation were detected by flow cytometry for cell surface markers, and the detection results showed (Table 4): MSC positive indicators CD90, CD73, CD105, CD44 were greater than 95%; negative indicators CD11B, CD19, CD31, CD34, CD45, HLADR were less than 2%, meeting the MSC attribute requirements.

[0133] Table 4

[0134]

[0135] The MSC cells differentiated by different passages (P13, P17, P21) of BC-hiPSC-ME-20 were detected for three-line (adipogenic, osteogenic, chondrogenic) differentiation. The results showed (Table 5): Figure 17 ), the MSC cells differentiated by different passages (P13, P17, P21) of BC-hiPSC-ME-20 all had adipogenic, osteogenic, chondrogenic differentiation ability, meeting the MSC attribute requirements.

[0136] 3. Directional induction and differentiation of BC-hiPSC-ME-20 into chondrocyte cells

[0137] BC-hiPSC-ME-20 was directionally differentiated into chondrocyte cell spheres according to the method in Example 1 of the patent CN118109397B.

[0138] The morphology of the chondrocyte cell spheres is shown in Figure 18 . The specific staining results are shown in Figure 19 . The detection results of the changes in the sGAG content (wet weight normalized sGAG content) of the key gene of the chondrocyte cell spheres are shown in Figure 20 . The results show that the sGAG content of the chondrocyte cell spheres gradually increased with the extension of the induction time, and remained stable at 3D46d.

[0139] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part 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 application.

Claims

1. Human induced pluripotent stem cells BC-hiPSC-ME-20, characterized in that, It is preserved in China General Microbiological Culture Collection Center, and the preservation number is 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 of the following stem cell markers: OCT4, NANOG, SOX2, TRA-1-81, SSEA-4, and 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 three germ layers, and has mesoderm-specific differentiation ability.

4. A progeny cell of the human induced pluripotent stem cell BC-hiPSC-ME-20 according to any one of claims 1 to 3.

5. A pharmaceutical composition or cell therapy agent, characterized in that, The pharmaceutical composition or cell therapeutic agent comprises 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) the progeny cell according to claim 4.

6. A medical device product, characterized by The medical device product comprises 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) the progeny cell according to claim 4.

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