Induced differentiation culture medium for mesenchymal stem cells and use thereof

Abstract

An induced differentiation culture medium for mesenchymal stem cells and a use thereof are provided. The mesenchymal stem cell-induced differentiation culture medium is used for directly inducing the differentiation of human induced pluripotent stem cells and / or embryonic stem cells into mesenchymal stem cells. The culture medium comprises: a mesenchymal stem cell basal culture medium and additives. The additives comprise the following components: 2-10 μM of a TGF-β inhibitor, 2-10 μM of a GSK3β inhibitor, and 5-10 μM of a ROCK inhibitor. By using the provided induced differentiation culture medium, human induced pluripotent stem cells and / or embryonic stem cells can be directly induced to differentiate into mesenchymal stem cells, and the obtained mesenchymal stem cells have the advantages of an unlimited source, stable quality, and high homogeneity. Furthermore, human induced pluripotent stem cells and / or embryonic stem cells can be directly and specifically differentiated into mesenchymal stem cells within a short period of time, thereby shortening the culture period and reducing production costs.

Description

A mesenchymal stem cell differentiation induction culture medium and its application Technical Field

[0001] This invention belongs to the field of stem cell biology and relates to lineage-specific differentiation of human pluripotent or embryonic stem cells, specifically to a mesenchymal stem cell induction differentiation culture medium and its preparation method. Background Technology

[0002] Mesenchymal stem cells (MSCs) can be isolated from various human tissues, such as bone marrow, adipose tissue, umbilical cord blood, peripheral blood, neonatal umbilical cord tissue, and placenta. However, the number of MSCs obtainable from adult tissues is limited, and invasive procedures are required for their isolation, which can pose unexpected risks to the donor. Obtaining a sufficient number of homogeneous and high-quality MSCs is crucial for the practical application of MSCs.

[0003] Human pluripotent stem cells include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). Pluripotent stem cells can proliferate indefinitely in vitro and maintain their potential to differentiate into almost all adult cells. Induced mesenchymal stem cells (iMSCs) can be obtained by directed differentiation of human pluripotent stem cells, providing a stable seed cell for MSC cell therapy. Currently, there are numerous reports on obtaining mesenchymal stem cells using this strategy, and compared to tissue-derived MSCs, iMSCs have advantages such as greater homogeneity, more stable biological properties, and predictable biological functions. However, the rarity of MSCs, their heterogeneity in different tissues and organs, and the need for invasive methods (except for umbilical cord-derived MSCs) limit their potential for clinical translation. Furthermore, MSCs have limited expansion capacity during culture, typically beginning to age after 8-10 passages, thus failing to produce sufficient cell numbers.

[0004] Therefore, finding simple, reliable, and sufficient cell sources is crucial for the application and transformation of MSCs. Induced pluripotent stem cells (iPSCs) are pluripotent stem cells with characteristics similar to embryonic stem cells, obtained by reprogramming adult cells. iPSCs can be cultured and expanded in vitro without restriction, continuously producing large quantities of adult stem cells for application. Therefore, iPSCs are gradually becoming the best cell source for obtaining MSCs. Currently, most established methods for differentiating various cell types, including MSCs, from iPSCs are based on the spontaneous differentiation of embryoid bodies (EBs). This type of method usually takes 30-40 days, is time-consuming, inefficient, and uncontrollable. There are also methods that induce pluripotent stem cells to first form trophoblast stem cells, and then further differentiate the trophoblast stem cells into mesenchymal stem cells. This type of method has problems such as incomplete shedding of upper cells, long processing time, mixed cells, insufficient differentiation, and low purity of mesenchymal stem cells. Meanwhile, most of the existing publicly available methods for preparing MSCs using iPSC-directed induction require adherent culture, digestion, and repeated passage, and involve co-culture of mouse cells, coating with xenogeneic animal-derived materials, flow cytometry sorting, or viral transfection. These methods are very complex and cumbersome to operate, and the yield is small and the quality is unstable, making them unsuitable for large-scale production. Furthermore, the introduction of exogenous xenogeneic materials during the culture process limits their application value.

[0005] Based on this, the present invention aims to provide a mesenchymal stem cell induction differentiation culture medium and its application. Summary of the Invention

[0006] The purpose of this invention is to provide a mesenchymal stem cell induction differentiation culture medium and its application.

[0007] According to a first aspect of the present invention, the present invention provides a mesenchymal stem cell induction differentiation medium, the mesenchymal stem cell induction differentiation medium being used to directly induce human induced pluripotent stem cells / or embryonic stem cells to differentiate into mesenchymal stem cells, the medium comprising: a mesenchymal stem cell basal medium and an additive, the additive comprising the following components: 2-10 μM TGF-β inhibitor, 2-10 μM GSK3β inhibitor and 5-10 μM Rock inhibitor.

[0008] Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the TGF-β inhibitor can be 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or any range of two of the above values, not limited to the listed values; other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the GSK3β inhibitor can be 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or any range of two of the above values, not limited to the listed values; other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the Rock inhibitor can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or any range of two of the above values, not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0009] In some embodiments of the present invention, the additive further comprises: human platelet lysate, ascorbic acid, GlutaMax additive, NEAA non-essential amino acids, ITS-X supplement, IGF, bFGF, and PDGF-BB.

[0010] In some embodiments of the present invention, the additive further comprises: human platelet lysate at a volume concentration of 1-10%, 30-70 mM ascorbic acid, GlutaMax additive at a volume concentration of 0.5%-2%, NEAA non-essential amino acids at a volume concentration of 0.5%-2%, ITS-X supplement at a volume concentration of 0.5%-2%, 1-10 ng / ml IGF, 1-10 ng / ml bFGF, and 1-10 ng / ml PDGF-BB.

[0011] Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of human platelet lysate, by volume concentration, can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range of any two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of ascorbic acid can be 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, or 50mM. M, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, or a range consisting of any two of the above values, not limited to the listed values; other unlisted values ​​within this range also apply. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of GlutaMax additive, by volume concentration, can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values, not limited to the listed values. Other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of the non-essential amino acid NEAA, by volume concentration, can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of ITS-X supplement, by volume concentration, can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable.Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of IGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, or a range of any two of the above values, and is not limited to the listed values; other values ​​within this range that are not listed are also applicable. Similarly, in the mesenchymal stem cell induction differentiation medium, the amount of bFGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, or a range of any two of the above values, and is not limited to the listed values; other values ​​within this range that are not listed are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of PDGF-BB can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0012] In some embodiments of the present invention, the TGF-β inhibitor is selected from at least one of SB431542, A-8301, and LY2157299.

[0013] In some embodiments of the present invention, the GSK3β inhibitor is selected from at least one of CHIR99021, SB415286, and LY2090314.

[0014] In some embodiments of the present invention, the Rock inhibitor is selected from at least one of Y-27632 and Blebbistatin.

[0015] In some embodiments of the present invention, the mesenchymal stem cell basal culture medium is selected from serum-free mesenchymal stem cell basal culture medium; the serum-free mesenchymal stem cell basal culture medium is selected from at least one of high glucose DMEM, Alpha-MEM, and DMEM / F12 culture medium.

[0016] According to a second aspect of the present invention, the present invention also provides a method for directly inducing human induced pluripotent stem cells / or embryonic stem cells to differentiate into mesenchymal stem cells, the method comprising: culturing human induced pluripotent stem cells / or embryonic stem cells in a mesenchymal stem cell induction differentiation medium as described in any of the first aspects of the present invention, and directly inducing the human induced pluripotent stem cells / or embryonic stem cells to differentiate into mesenchymal stem cells.

[0017] In some embodiments of the present invention, the method includes:

[0018] 1. Provide cell cultures of human pluripotent stem cells or embryonic stem cells;

[0019] 2. Replace the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium and perform passage culture to obtain P3-P5 generation mesenchymal stem cells or cell cultures including P3-P5 generation mesenchymal stem cells.

[0020] In some embodiments of the present invention, step one, the cell culture step includes: culturing the human pluripotent stem cells or embryonic stem cells in a culture medium without xenogeneic animal-derived components, using a culture method without feeder layer cells; preferably, the culture step includes first culturing the human pluripotent stem cells or embryonic stem cells normally in a maintenance medium until the cell confluence reaches 80-90%, digesting the iPSC cells into a complete single-cell suspension using Tryple, resuspending it in iPSC maintenance medium, adding a Rock inhibitor to the iPSC maintenance medium, maintaining the Rock inhibitor for 24 hours, and then replacing it with complete iPSC maintenance medium to obtain the cell culture with a confluence of 30%-50%.

[0021] In some embodiments of the present invention, the maintenance culture medium is selected from at least one of E8, StemFit Basic04, and mTeSR Plus.

[0022] In some embodiments of the present invention, the Rock inhibitor is selected from at least one of Y-27632 and Blebbistatin.

[0023] In some embodiments of the present invention, the substrate gel used in the culture process is selected from at least one of Laminin-521, Vitronectin, and Matrigel.

[0024] In some embodiments of the present invention, the culture conditions used in the culture process include: 35-40°C, 4-6% CO2, and 92-98% humidity.

[0025] In some embodiments of the present invention, step two, which involves replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium for passage culture, includes the following steps: replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium, culturing until the cell confluence reaches 80%-90% (recorded as P0 generation), digesting the cells, resuspending the digested cells in the mesenchymal stem cell induction differentiation medium, and continuing to culture to obtain P1 generation mesenchymal stem cells, repeating the above passage culture steps to obtain P3-P5 generation mesenchymal stem cells.

[0026] In some embodiments of the present invention, the digestion process includes: removing the supernatant, washing the cells with calcium- and magnesium-free DPBS, digesting the washed cells, centrifuging to remove the supernatant, and obtaining the digested cells.

[0027] In some embodiments of the present invention, the method further includes: placing the mesenchymal stem cells or cell cultures including mesenchymal stem cells in a mesenchymal stem cell expansion culture medium for expansion and passage culture to obtain the mesenchymal stem cells.

[0028] In some embodiments of the present invention, the mesenchymal stem cell expansion culture medium comprises: mesenchymal stem cell basal culture medium and additives, wherein the additives further comprise: human platelet lysate, ascorbic acid, GlutaMax additive, NEAA non-essential amino acids, ITS-X supplement, IGF, bFGF, and PDGF-BB.

[0029] In some embodiments of the present invention, the additive further comprises: human platelet lysate at a volume concentration of 1-10%, 30-70 mM ascorbic acid, GlutaMax additive at a volume concentration of 0.5%-2%, NEAA non-essential amino acids at a volume concentration of 0.5%-2%, ITS-X supplement at a volume concentration of 0.5%-2%, 1-10 ng / ml IGF, 1-10 ng / ml bFGF, and 1-10 ng / ml PDGF-BB.

[0030] Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of human platelet lysate, by volume concentration, can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range of any two of the above values, and is not limited to the listed values; other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of ascorbic acid can be 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, or 51mM. The range is 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, or any two of the above values. It is not limited to the listed values; other unlisted values ​​within this range also apply. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of GlutaMax additive, by volume concentration, can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values, not limited to the listed values. Other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of the non-essential amino acid NEAA, by volume concentration, can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of ITS-X supplement, by volume concentration, can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable.Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of IGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, or a range of any two of the above values, and is not limited to the listed values; other values ​​within this range that are not listed are also applicable. Similarly, in the mesenchymal stem cell induction differentiation medium, the amount of bFGF can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, or a range of any two of the above values, and is not limited to the listed values; other values ​​within this range that are not listed are also applicable. Specifically, in the mesenchymal stem cell induction differentiation culture medium, the amount of PDGF-BB can be 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, or a range of any two of the above values. It is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0031] In some embodiments of the present invention, the mesenchymal stem cell basal culture medium is selected from serum-free mesenchymal stem cell basal culture medium; the serum-free mesenchymal stem cell basal culture medium is selected from at least one of high glucose DMEM, Alpha-MEM, and DMEM / F12 culture medium.

[0032] According to a third aspect of the present invention, the present invention also provides mesenchymal stem cells obtained based on any of the methods described in any of the second aspects of the present invention.

[0033] In some embodiments of the present invention, the mesenchymal stem cells are pluripotent cells capable of differentiating into adipocytes, osteocytes, chondrocytes, myocytes, nerve cells, and cardiomyocytes.

[0034] In some embodiments of the present invention, the mesenchymal stem cells are able to express cell surface markers such as CD29, CD73, CD90, CD105, CD166, and CD44.

[0035] In some embodiments of the present invention, the mesenchymal stem cells do not express cell surface markers such as HLA-DR, CD34, CD45, CD19, and CD14.

[0036] In some embodiments of the present invention, the mesenchymal stem cells are P5 generation or higher mesenchymal stem cells, and the proportion of cells expressing CD29, CD44, CD73, CD90, CD105, and CD166 in the mesenchymal stem cells is not less than 95%. In some embodiments of the present invention, the proportion of cells expressing HLA-DR, CD34, CD45, CD19, and CD14 in the mesenchymal stem cells is not more than 2%.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) This application provides a culture medium for directly inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells and a method for directly inducing human induced pluripotent stem cells to differentiate into mesenchymal stem cells. The induction differentiation culture medium provided in this application can directly induce human induced pluripotent stem cells to differentiate into mesenchymal stem cells. The cell differentiation pathway is clear, the differentiation efficiency is high, and the differentiation effect is stable. No culture system or feeder cells containing serum are used. The obtained cell population has high purity and large quantity. It solves the problems of long induction differentiation time, long passage time and slow cell proliferation in the prior art. The serum-free and xenogeneic culture system solves the safety problem. Even after repeated passage culture, the characteristics of mesenchymal stem cells can be maintained for a long time.

[0039] (2) This invention significantly improves the differentiation efficiency of mesenchymal stem cells (MSCs) and maintains their excellent characteristics even after long-term passage culture (e.g., 12, 15, or more passages). This passage method allows for the large-scale preparation of human pluripotent stem cell-derived MSCs. During differentiation, the differentiation pathway is precisely controlled through the combined use of small molecule compounds, achieving stable and efficient differentiation, ultimately yielding mature MSCs. The surface factor expressed by the obtained MSCs is CD90. + CD73 + CD105 + CD14 - CD34 - CD45 - CD19 - and HLA-DR - It adheres to the cell wall and has the ability to differentiate into bone and fat, without the need for additional flow cytometry screening technology to select cells.

[0040] (3) Compared with the existing technology, which discloses the induction method of multipotent stem cells first forming trophoblast stem cells and then differentiating into mesenchymal stem cells, the upper layer cells are easy to detach, the process is incomplete and time-consuming, there are many mixed cells and insufficient differentiation, and the purity of mesenchymal stem cells is low. The induction differentiation medium provided in this application can directly induce human induced pluripotent stem cells to differentiate into mesenchymal stem cells. This application is time-efficient and constructs an induction differentiation system with clearly defined components. The serum-free culture system efficiently induces differentiation into mesenchymal stem cells of uniform purity and stable performance. Compared to the spontaneous differentiation of embryoid bodies (EBs) disclosed in the prior art, which suffers from low induction efficiency, easy retention of other pluripotent cells, time-consuming, labor-intensive, inefficient, and uncontrollable problems, the induction differentiation medium provided in this application simplifies the experimental procedure and has good reproducibility. In other words, compared to the existing methods that first form embryoid bodies (EBs) for spontaneous differentiation or induce pluripotent stem cells to first form trophoblast stem cells and then differentiate into mesenchymal stem cells, the method provided in this application is time-efficient and efficiently induces differentiation into uniform purity and stable performance using a serum-free culture system with clearly defined components. Attached Figure Description

[0041] Figure 1 shows the pluripotency identification results of iPSC cells in Example 1 of this application;

[0042] Figure 2 shows the cell morphology of iMSCs derived from iPSCs at different passages obtained in Example 2 of this application;

[0043] Figure 3A shows the CD90 of iMSCs detected by flow cytometry in the example of the effect of this application. + CD73 + CD105 + CD45 - The proportion of each group;

[0044] Figure 3B shows the flow cytometry detection of CD14 in iMSCs in the example of the effect of this application. - CD34 - CD19 - and HLA-DR - The proportion of phenotypes;

[0045] Figure 4 shows the results of RT-QPCR detection of the expression of MSC-related marker genes OCT4, SNAI2, COL6A2, and TWIST1 in the effect example of this application;

[0046] Figure 5 shows the results of detecting the expression of osteogenic specific genes OCN, ALP, and RUNX2 in iMSC in the effect example of this application;

[0047] Figure 6 shows the results of the osteogenic differentiation capacity of iMSC in the example of the application;

[0048] Figure 7 shows the results of detecting the expression of adipogenic specific genes PPARr2 and LPL in iMSC in the effect example of this application;

[0049] Figure 8 shows the detection results of the adipogenic differentiation capacity of iMSC in the effect example of this application;

[0050] Figure 9 shows the cell culture status of different induction differentiation media 1-6 in the comparative examples of this application;

[0051] Figure 10 shows the cell state of iPSC-iMSCs in groups A, B, and C of the comparative examples of this application on day 6 after differentiation in the induction differentiation medium. Detailed Implementation

[0052] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0053] The cell and reagent information used in the embodiments of this invention is as follows:

[0054] Induced pluripotent stem cells: Product code: hCiPSC-00409, Manufacturer: Beijing Beiqi Biomedical Co., Ltd.;

[0055] Laminin-521 stock solution: Product No.: 200-0117, Manufacturer: Stemcell;

[0056] DPBS (containing calcium and magnesium): Product No.: 14080055, Manufacturer: Gibco;

[0057] DPBS (calcium and magnesium-free): Product number: C14190500BT, Manufacturer: Gibco;

[0058] Vitronectin stock solution: Product No.: RP01002, Manufacturer: Shouning Biotechnology;

[0059] DMEM / F12: Part No.: 11330-032, Manufacturer: Gibco;

[0060] mTeSR Plus culture medium: Catalog number: 100-0276, Manufacturer: Stemcell;

[0061] TrypLE TM Express: Item No.: 12604021, Manufacturer: Gibco;

[0062] Y27632: Part No.: HY-10071, Manufacturer: MCE;

[0063] Versene digestive enzymes: Product code: 15040066; Manufacturer: Gibco;

[0064] Human platelet lysate: Catalog number: PL-NH-100, Manufacturer: Sexton;

[0065] Ascorbic acid: Product code: A8960, Manufacturer: Merck;

[0066] GlutaMax Additive: Product No.: A1286001, Manufacturer: Gibco;

[0067] NEAA (Non-essential Amino Acids): Product No.: 11140050, Manufacturer: Gibco;

[0068] ITS-X supplement: Product number: 51500056, Manufacturer: Gibco;

[0069] IGF: Product No.: GMP-C023, Manufacturer: Nearshore Protein;

[0070] bFGF: Product No.: GMP-C046, Manufacturer: Nearshore Protein;

[0071] PDGF-BB: Product code: GMP-C199, Manufacturer: Nearshore Protein.

[0072] Example 1: Culture and passage of initial cells

[0073] In this embodiment, iPSC cells were used as the initial cells, as shown in Figure 1. According to the results in Figure 1, iPSC cells exhibited typical clonal cluster growth characteristics: clear clonal edges, close cell contact within the clone, high nucleo-cytoplasmic ratio, homogeneous morphology, and no differentiated cells were observed. The iPSC cell culture and passage procedures are as follows:

[0074] Laminin-521 coating culture plate: Take the Laminin-521 stock solution and dilute it to a final concentration of 5 μg / mL using DPBS (containing calcium and magnesium); add the diluted Laminin-521 to the culture plate and place it in a refrigerator at 2-8℃ overnight or at 37℃ for at least 2 hours for coating. After coating, take it out of the refrigerator 30 minutes before use and place it at room temperature for later use.

[0075] Vitronectin coating culture plate: Take Vitronectin stock solution and dilute it to a final concentration of 10 μg / mL using DMEM / F12; add the diluted Vitronectin to the culture plate and place it in a refrigerator at 2-8℃ overnight or at room temperature for at least 1 hour for coating. After coating, take it out of the refrigerator 30 minutes before use and place it at room temperature for later use.

[0076] Cell resuscitation: Transfer the thawed iPSC cell suspension to a new 15mL centrifuge tube, and gently add room temperature-equilibrated human induced pluripotent stem cell complete culture medium dropwise to the cell suspension while gently agitating the centrifuge tube. Transfer the 15mL centrifuge tube containing the iPSC cell suspension to a low-speed refrigerated centrifuge and centrifuge at 300g for 3 minutes at room temperature. After centrifugation, discard the supernatant, add 10μM Y27632 human induced pluripotent stem cell complete culture medium (mTeSR Plus) to resuspend the cells, and gently pipette them into preheated 12Well culture plates at a volume ratio of 1:15. Incubate at 37℃, 5% CO2, and 95% humidity. Change the medium every 20-24 hours. When the cell coverage reaches more than 80%, begin cell passage.

[0077] Cell passage: Once the cell coverage reaches 80% or more, remove the cell culture plate from the incubator and wash the cells once with DPBS (calcium and magnesium-free). Then, add Versene digestive enzyme to the culture plate to digest the cells for 3-5 minutes. Observe the cell colony dissociation state under a microscope. After the cells are completely dissociated, add human induced pluripotent stem cell complete culture medium (mTeSR Plus) and gently pipette the cell suspension. Take out another blank culture plate preheated to 37°C from the incubator and add the cell suspension at a volume ratio of 1:20. Incubate overnight at 37°C with 5% CO2. Change the cell medium every 20-24 hours. When the cell coverage reaches 80% or more, start the next cell passage. Passage the cells to P20 in the above manner to obtain P20 generation cells.

[0078] Example 2

[0079] This embodiment provides a method for preparing human induced pluripotent stem cells (iPSCs) by directly inducing their differentiation into mesenchymal stem cells, comprising the following steps:

[0080] I. iPSC Culture: The iPSC cells passaged to the P20 generation obtained in Example 1 were cultured normally in maintenance medium, specifically mTeSR Plus. When the iPSC cells reached a confluence of 80-90%, they were digested into a complete single-cell suspension using Tryple and resuspended in iPSC maintenance medium. A Rock inhibitor was added to the iPSC maintenance medium, and after 24 hours of maintenance with the Rock inhibitor, the medium was replaced with complete iPSC maintenance medium. The iPSCs were cultured using 12 wells, with mTeSR Plus as the maintenance medium and Y-27632 as the Rock inhibitor at a concentration of 10 μM. The confluence of the cultured iPSC cells was 30%-50%.

[0081] II. Induction of iPSC cells into mesenchymal stem cells (MSCs):

[0082] Replace the maintenance medium for iPSC cells in step one with mesenchymal stem cell induction differentiation medium, and incubate in a 5% CO2, 37°C incubator. Record this cell type as P0. When cell confluence reaches 80%-90%, aspirate the supernatant, wash the cells twice with pre-warmed calcium- and magnesium-free DPBS, then digest the cells with pre-warmed Tryple until they are in a single-cell state. Stop digestion, centrifuge, remove the supernatant, and resuspend the cells in 1 mL of mesenchymal stem cell induction differentiation medium. Repeat the induction process at 5 × 10⁻⁶ cells / mL. 4 cells / cm 2 The cells were seeded at a density into wells pre-coated with Laminin-521 and cultured in a 5% CO2, 37°C incubator. This initial cell culture was designated P1. Following the same steps, the cells were passaged to generation P3. The mesenchymal stem cell induction differentiation medium consisted of: serum-free basal medium for mesenchymal stem cells (selected from DMEM / F12 medium), containing 5% (v / v) human platelet lysate, 50 mM ascorbic acid, 1% (v / v) GlutaMax additive, 1% (v / v) NEAA non-essential amino acids, 1% (v / v) ITS-X supplement, 5 ng / ml IGF, 5 ng / ml bFGF, 5 ng / ml PDGF-BB, and 5 μM TGF-β inhibitor (A8301), 5 μM GSK3β inhibitor (CHIR99021), and 8 μM Rock inhibitor (Y-27632).

[0083] III. iMSC Amplification and Passaging

[0084] Once the P3 generation cells obtained in step two above have reached approximately 80%-90% confluence, re-digest the cells until they are in a single-cell state. Resuspend the cells in 1 mL of mesenchymal stem cell expansion culture medium at a concentration of 0.9 × 10⁻⁶. 4 cells / cm 2 The cells were seeded at a density in pre-coated Laminin-521-coated wells and cultured in a 5% CO2, 37°C incubator for 3-5 days. Once the cell confluence reached approximately 80%-90%, the cells were passaged in the same manner as described above until passage P12, yielding P3-P12 generation mesenchymal stem cells. The mesenchymal stem cell expansion medium consisted of: serum-free basal medium for mesenchymal stem cells (selected from DMEM / F12 medium), containing 5% (v / v) human platelet lysate, 50 mM ascorbic acid, 1% (v / v) GlutaMax additive, 1% (v / v) NEAA non-essential amino acids, 1% (v / v) ITS-X supplement, 5 ng / ml IGF, 5 ng / ml bFGF, and 5 ng / ml PDGF-BB.

[0085] Example 1

[0086] The morphology of the P3 generation and subsequent passaged mesenchymal stem cells prepared in Example 2 was observed. The results are shown in Figure 2. The cells from P3 to P12 all exhibited the parallel spiral MSC morphology characteristics. No senescence phenomena such as increased cell volume or slowed proliferation were observed, and they were able to proliferate and pass on stably.

[0087] Meanwhile, the P8 generation mesenchymal stem cells prepared in Example 2 were collected and analyzed by flow cytometry. The results are shown in Figure 3. The corresponding mesenchymal stem cells showed CD73... + CD90 + and CD105 + The expression rate of CD19 was over 95%, indicating a positive result. - CD14 - CD34 - CD45 - and HLA-DR - Expression of less than 2% is considered negative. It should be noted that although this invention only tested flow cytometry results of P8 generation cells in this efficacy experiment, according to the latest experimental data, the optimal range is up to P12 generation.

[0088] Example 2

[0089] To verify the effect of ipsc in the mesenchymal stem cell induction culture medium provided by this invention on the gradual differentiation into iMSCs, this effect was further verified by detecting the expression of relevant genes in the relevant cells. The specific steps are as follows:

[0090] The experiment was divided into three groups: iMSC group (P1, P3, P5, and P7 generation mesenchymal stem cells prepared in Example 2 of this invention), iPSC group (prepared in Example 1 of this invention), and ADSC group (adipose-derived mesenchymal stem cells). RNA was extracted from the cells of the above three groups, and the expression of OCT4, TWIST1, COL6A2, and SNAI2 was detected by real-time quantitative RT-QPCR. The specific steps are as follows:

[0091] (1) RNA extraction: RNA was extracted using an RNA purification kit (manufacturer: TransGen, catalog number: ER101-01);

[0092] (2) Reverse transcription: Take 1 μg of the prepared RNA sample and perform reverse transcription according to the requirements of the chain reaction reverse transcription reagent (manufacturer: TransGen, catalog number: AU341-02). The reverse transcription system is as follows:

[0093] After mixing the above samples, centrifuge, incubate at 42℃ for 15 min, inactivate at 85℃ for 5 s, and dilute the obtained cDNA by 2 times for subsequent reactions.

[0094] (3) Quantitative Real-time PCR (qRT-PCR): The samples obtained above were subjected to qRT-PCR detection according to the following system:

[0095] The detection results obtained through the above method are shown in Figure 4. According to the results shown in Figure 4, the mesenchymal stem cells prepared at different passage numbers showed significant expression of TWIST1, COL6A2, and SNAI2. The gene detection results of P1, P3, P5, and P7 generation cells shown in Figure 4 indicate that the cells have clearly transitioned from epithelial to mesenchymal (EMT) morphology. Combined with the flow cytometry results of P8 generation cells shown in Figure 3, this further confirms that the cells are iMSC cells. OCT4 is a marker gene for iPSCs. When iPSCs successfully differentiate into iMSCs, each generation of cells does not express OCT4, as shown in Figure 4. P1, P3, P5, and P7 generation cells express almost no OCT4 or only trace amounts, further demonstrating that the differentiation induction method provided by this invention can successfully induce iPSCs to differentiate into iMSCs.

[0096] Example 3

[0097] The osteogenic and adipogenic differentiation capabilities of the P8 generation mesenchymal stem cells prepared in Example 2 were further tested, as follows:

[0098] (1) Identification of osteogenic differentiation:

[0099] P8 generation mesenchymal stem cells were seeded into cell culture containers at an appropriate seeding density. An appropriate amount of preheated fresh iMSC expansion medium was added, and the mixture was shaken horizontally three times. The containers were then placed in an incubator at 37°C, 5% CO2 concentration, and saturated humidity. The mixture was shaken horizontally three times again and cultured. When the iMSCs spread evenly and reached a confluence of 80%-90%, the medium in the container was aspirated and replaced with osteogenic differentiation induction medium, designated as day 0. The medium was completely replaced every three days and cultured until day 21. The osteogenic differentiation induction culture used was Alpha-MEM medium containing 10% FBS, 1% Penicillin-Streptomycin, 10 mM β-glycerophosphate, 10 nM Dexamethasone, and 50 μg / ml Ascorbic Acid.

[0100] RNA was observed and photographed under a light microscope and collected from differentiated cells cultured to day 4, day 14, and day 21. The expression of osteogenic-specific genes RUNX2, ALP, and OCN was tested, and the results are shown in Figure 5. According to the results shown in Figure 5, with the increase of differentiation culture time, the expression levels of specific genes ALP and OCN gradually increased, while the expression level of specific gene RUNX2 first decreased and then increased, indicating that the differentiated iMSCs have the ability to differentiate into osteoblasts. After day 21, the osteogenic differentiated iMSCs were washed with pure water, and an appropriate volume of alizarin red working solution was added. They were incubated at room temperature in the dark for 20-30 minutes, and then the excess staining solution was aspirated. An appropriate volume of physiological saline or DPBS was added to each well for infiltration. The cells were observed and photographed under a microscope, and the results are shown in Figure 6. According to the results shown in Figure 6, calcium nodules were clearly formed.

[0101] (2) Identification of adipogenic differentiation:

[0102] P8 generation mesenchymal stem cells were seeded into cell culture containers at an appropriate seeding density, and an appropriate amount of preheated fresh iMSC expansion medium was added. The containers were then shaken horizontally three times and placed in an incubator at 37°C, 5% CO2 concentration, and saturated humidity. The containers were shaken horizontally three more times and cultured. Once the iMSCs had grown evenly and reached 80%-90% confluence, the culture medium was aspirated from the containers and replaced with adipogenic differentiation induction medium, designated as day 0. The medium was completely replaced every three days, and cultured until day 21. The adipogenic differentiation induction medium used was Alpha-MEM medium containing 10% FBS, 1% Penicillin-Streptomycin, 1 μM Dexamethasone, 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 0.2 mM Indomethacin, and 10 ug / ml insulin.

[0103] RNA was observed and photographed under a light microscope from differentiated cells cultured to day 4, day 14, and day 21 to test the expression of adipogenic-specific genes PPARr2 and LPL. The results are shown in Figure 7. According to Figure 7, with increasing differentiation culture time, the expression level of PPARr2 showed a trend of first increasing and then decreasing, while the expression level of LPL showed a gradual increasing trend, indicating that differentiated iMSCs have the ability to differentiate into adipocytes. Meanwhile, during normal differentiation, cells gradually widened and shortened, and many round fat granules could be seen within the cells under high magnification. After Day 21, the adipogenic differentiated iMSCs were washed with physiological saline or DPBS, and then washed with 60% isopropanol to prevent residual physiological saline or DPBS from causing staining solution precipitation. An appropriate volume of Oil Red O working solution was added to the differentiation group and the control group, and the cells were incubated at room temperature in the dark for 20-60 min. Then, excess staining solution was aspirated, and the cells were washed with physiological saline or DPBS until no background color was visible. An appropriate volume of physiological saline or DPBS was added to each well for infiltration, and the cells were observed and photographed under a microscope. The results are shown in Figure 8. According to the results shown in Figure 8, lipid droplets were clearly formed.

[0104] Comparative Example

[0105] This comparative study further investigated the effects of different induction differentiation media on the properties of the resulting mesenchymal stem cells, as detailed below:

[0106] The following groups are set up respectively:

[0107] Culture medium #1: iMSC amplification medium;

[0108] Medium 2: Add 5 μM of GSK3β inhibitor (CHIR99021) to the iMSC amplification medium;

[0109] Culture medium #3: iMSC amplification medium with 5 μM TGF-β inhibitor (A8301) added;

[0110] Culture medium #4: iMSC amplification medium with 8 μM of Rock inhibitor (Y-27632) added;

[0111] Medium No. 5: iMSC amplification medium supplemented with 5 μM GSK3β inhibitor (CHIR99021) and 5 μM TGF-β inhibitor (A8301);

[0112] Medium No. 6: iMSC amplification medium supplemented with 5 μM GSK3β inhibitor (CHIR99021), 5 μM TGF-β inhibitor (A8301), and 8 μM Rock inhibitor (Y-27632);

[0113] The iMSC amplification medium formula is as follows: serum-free basal medium for mesenchymal stem cells (basal medium selected from DMEM / F12 medium), containing 5% human platelet lysate, 50mM ascorbic acid, 1% GlutaMax additive, 1% NEAA non-essential amino acids, 1% ITS-X supplement, 5ng / ml IGF, 5ng / ml bFGF, and 5ng / ml PDGF-BB.

[0114] After passage, the iPSCs prepared in Example 1 were aspirated from the old medium and divided into 8 portions. Each portion was then replaced with one of the 8 different culture media mentioned above, and the cells were cultured in a 5% CO2, 37°C incubator. The corresponding fresh induction differentiation medium was replaced daily, and cell morphology and proliferation were observed. Cells were passaged when the confluence reached 85%–90%. Cell morphology was observed during the differentiation of iPSCs into iMSCs, and the results are shown in Figure 9. According to Figure 9, the cell morphology in medium 1 remained largely unchanged, and cell proliferation was minimal. Cells in medium 2 gradually died during differentiation. Cells in medium 3 showed largely unchanged morphology. Cells in media 4 and 5 showed no significant proliferation when passaged to the P1 generation. Cells in medium 6, when passaged to the P2 generation, adhered normally, and most cells exhibited a short spindle shape.

[0115] Further comparisons were made regarding the effects of inducing differentiation of iPSCs using the following three groups of induction culture media on the preparation of mesenchymal stem cells, as detailed below:

[0116] Group A culture medium: Culture medium No. 6;

[0117] Group B culture medium: basal medium E6 (brand: Gibco, catalog number: A1516401), supplemented with 10 μM TGF-β inhibitor (SB431542), 2 μM GSK3β inhibitor (CHIR99021), and 10 ng / ml bFGF;

[0118] Group C culture medium: basal medium E6 (brand: Gibco, catalog number: A1516401), supplemented with 0.1 mM 2-mercaptoethanol, 1% ITS-X supplement, 1.5 μg / ml L-AA-pi, 50 ng / ml EGF (epidermal growth factor), 2 μM GSK3β inhibitor (CHIR99021), 0.5 μM TGF-β inhibitor (A83-01), 1 μM TGF-β inhibitor (SB431542), 0.8 mM VPA (valproic acid), 5 μM Rock inhibitor (Y27632) and 10 ng / mL BMP4;

[0119] The iPSC single cells prepared in Example 1 were seeded and cultured for 2 days. When the cell confluence reached about 30%, they were divided into 3 groups and the iPSC-iMSC induction differentiation medium for groups A, B, and C was changed respectively. The corresponding fresh induction differentiation medium was changed every day. The cell morphology was observed on day 6. The results are shown in Figure 10. According to the results shown in Figure 10, the cells in group A corresponding to medium 6 were in good differentiation state, and mesenchymal-like cells were observed under the microscope. However, in groups B and C, cell death occurred to varying degrees on day 6.

[0120] The results of cell morphology and proliferation shown in Figures 9 and 10 further demonstrate that direct differentiation culture of iPSCs using the induction differentiation medium provided by this invention can yield mesenchymal stem cells with excellent performance in all aspects.

[0121] It is understood that this invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of this invention. Furthermore, under the teachings of this invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of this invention. Therefore, this invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this invention are within the protection scope of this invention.

Claims

1. A mesenchymal stem cell induction differentiation culture medium, characterized in that, The mesenchymal stem cell induction differentiation medium is used to directly induce human induced pluripotent stem cells / or embryonic stem cells to differentiate into mesenchymal stem cells. The medium contains: a mesenchymal stem cell basal medium and an additive. The additive contains the following components: 2-10 μM TGF-β inhibitor, 2-10 μM GSK3β inhibitor and 5-10 μM Rock inhibitor.

2. The mesenchymal stem cell induction differentiation culture medium according to claim 1, characterized in that, The additives also include: human platelet lysate, ascorbic acid, GlutaMax additive, NEAA non-essential amino acids, ITS-X supplement, IGF, bFGF, and PDGF-BB; Preferably, the additive further comprises: human platelet lysate at a volume concentration of 1-10%, 30-70 mM ascorbic acid, GlutaMax additive at a volume concentration of 0.5%-2%, NEAA non-essential amino acids at a volume concentration of 0.5%-2%, ITS-X supplement at a volume concentration of 0.5%-2%, 1-10 ng / ml IGF, 1-10 ng / ml bFGF, and 1-10 ng / ml PDGF-BB.

3. The mesenchymal stem cell induction differentiation culture medium according to claim 1, characterized in that, The TGF-β inhibitor is selected from at least one of SB431542, A-8301, and LY2157299; the GSK3β inhibitor is selected from at least one of CHIR99021, SB415286, and LY2090314; and the Rock inhibitor is selected from at least one of Y-27632 and Blebbistatin.

4. The mesenchymal stem cell induction differentiation culture medium according to claim 1, characterized in that, The mesenchymal stem cell basal culture medium is selected from serum-free mesenchymal stem cell basal culture medium; the serum-free mesenchymal stem cell basal culture medium is selected from at least one of high glucose DMEM, Alpha-MEM, and DMEM / F12 culture medium.

5. A method for directly inducing human induced pluripotent stem cells / or embryonic stem cells to differentiate into mesenchymal stem cells, characterized in that, The method includes: passage culture of human induced pluripotent stem cells / or embryonic stem cells using the mesenchymal stem cell induction differentiation medium as described in any one of claims 1-4, and directly inducing the human induced pluripotent stem cells / or embryonic stem cells to differentiate into mesenchymal stem cells.

6. The method according to claim 5, characterized in that, The method includes:

1. Provide cell cultures of human pluripotent stem cells or embryonic stem cells; 2. Replace the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium and perform passage culture to obtain P3-P5 generation mesenchymal stem cells or cell cultures including P3-P5 generation mesenchymal stem cells.

7. The method according to claim 6, characterized in that, In step one, the cell culture process includes: culturing the human pluripotent stem cells or embryonic stem cells in a culture medium without xenogeneic animal-derived components using a feeder-free cell culture method; preferably, the culture process includes first culturing the human pluripotent stem cells or embryonic stem cells normally in a maintenance medium until the cells reach a confluence of 80-90%, then digesting the iPSC cells into a complete single-cell suspension using Tryple, resuspending it in iPSC maintenance medium, adding a Rock inhibitor to the iPSC maintenance medium, maintaining the Rock inhibitor for 24 hours, and then replacing it with complete iPSC maintenance medium to obtain the cell culture with a confluence of 30%-50%. Preferably, the maintenance culture medium is selected from at least one of E8, StemFit Basic04, and mTeSR Plus; Preferably, the Rock inhibitor is selected from at least one of Y-27632 and Blebbistatin; Preferably, the substrate gel used in the culture process is selected from at least one of Laminin-521, Vitronectin, and Matrigel; Preferably, the culture conditions include: culture in a constant temperature incubator at 35-40℃, 4-6% CO2, and 92-98% humidity.

8. The method according to claim 6, characterized in that, Step two, replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium for passage culture, includes the following steps: replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium, culturing until the cell confluence reaches 80%-90%, recorded as P0 generation, digesting the cells, resuspending the digested cells in the mesenchymal stem cell induction differentiation medium, and continuing to culture to obtain P1 generation mesenchymal stem cells, repeating the above passage culture steps to obtain P3-P5 generation mesenchymal stem cells; Preferably, the digestion process includes: removing the supernatant, washing the cells with calcium- and magnesium-free DPBS, digesting the washed cells, centrifuging to remove the supernatant, and obtaining the digested cells.

9. The method according to claim 6, characterized in that, The method further includes: placing the P3-P5 generation mesenchymal stem cells or cell cultures including P3-P5 generation mesenchymal stem cells in a mesenchymal stem cell expansion medium for expansion and passage culture to obtain the mesenchymal stem cells. Preferably, the mesenchymal stem cell expansion culture medium comprises: mesenchymal stem cell basal culture medium and additives, wherein the additives further comprise: human platelet lysate, ascorbic acid, GlutaMax additive, NEAA non-essential amino acids, ITS-X supplement, IGF, bFGF, and PDGF-BB. Preferably, the composition includes 1-10% human platelet lysate, 30-70 mM ascorbic acid, 0.5%-2% GlutaMax additive, 0.5%-2% NEAA non-essential amino acids, 0.5%-2% ITS-X supplement, 1-10 ng / ml IGF, 1-10 ng / ml bFGF, and 1-10 ng / ml PDGF-BB. Preferably, the mesenchymal stem cell basal culture medium is selected from serum-free mesenchymal stem cell basal culture medium; the serum-free mesenchymal stem cell basal culture medium is selected from at least one of high glucose DMEM, Alpha-MEM, and DMEM / F12 culture medium.

10. A mesenchymal stem cell prepared by the method according to any one of claims 5-9; Preferably, the mesenchymal stem cells are pluripotent cells capable of differentiating into adipocytes, osteocytes, chondrocytes, myocytes, nerve cells, and cardiomyocytes; Preferably, the mesenchymal stem cells are able to express cell surface markers such as CD29, CD73, CD90, CD105, CD166, and CD44; Preferably, the mesenchymal stem cells do not express cell surface markers such as HLA-DR, CD34, CD45, CD19, and CD14; Preferably, the mesenchymal stem cells are P5 generation or higher mesenchymal stem cells, and the proportion of cells expressing CD29, CD44, CD73, CD90, CD105, and CD166 in the mesenchymal stem cells is not less than 95%; preferably, the proportion of cells expressing HLA-DR, CD34, CD45, CD19, and CD14 in the mesenchymal stem cells is not more than 2%.

Images