Mesenchymal stem cell induced differentiation culture medium and application thereof

Abstract

The invention belongs to the field of stem cell biology, and relates to pedigree specific differentiation of human pluripotent or embryonic stem cells, in particular to a mesenchymal stem cell induced differentiation culture medium and application thereof. The mesenchymal stem cell induced differentiation culture medium is used for directly inducing and differentiating human induced pluripotent stem cells / or embryonic stem cells into mesenchymal stem cells, the culture medium comprises a mesenchymal stem cell basic culture medium and an additive, and the additive comprises the following components: 2-10 [mu] M of a TGF-beta inhibitor, 2-10 [mu] M of a GSK3 beta inhibitor and 5-10 [mu] M of a Rock inhibitor. By adopting the induced differentiation culture medium provided by the invention, human induced pluripotent stem cells / or embryonic stem cells can be directly induced and differentiated into mesenchymal stem cells, and the obtained mesenchymal stem cells have the advantages of infinite sources, stable quality, high homogeneity and the like; and the human induced pluripotent stem cells / or embryonic stem cells can be directly and directionally differentiated into the mesenchymal stem cells in a relatively short time, so that the culture time is shortened, and the production cost is reduced.

Description

Technical Field

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

[0002] Mesenchymal stem cells (MSC) can be isolated from various human tissues, such as bone marrow, adipose tissue, umbilical cord blood, peripheral blood, neonatal tissue umbilical cord, placenta, etc., but the number of mesenchymal stem cells that can be obtained from adult tissues is limited, and invasive procedures are required to isolate mesenchymal stem cells, which may bring unexpected risks to the donor. Obtaining sufficient number of uniform and stable quality MSC cells is the key to realizing the application of MSC cells.

[0003] Human pluripotent stem cells include human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPSC). Pluripotent stem cells can proliferate indefinitely in vitro and maintain their potential to differentiate into almost all adult cells. Induced mesenchymal stem cells (iMSC) can be obtained by induced differentiation of human pluripotent stem cells, which can provide a stable seed cell for MSC cell therapy. At present, there are many reports on the use of this strategy to obtain mesenchymal stem cells, and compared with tissue-derived MSCs, iMSCs have the advantages of more homogeneity, more stable biological properties and predictable biological functions. However, the rarity of MSCs, heterogeneity in different tissues and organs, and the need to obtain them by invasive means (except umbilical cord sources) limit the potential of MSCs for clinical transformation. In addition, the expansion capacity of MSCs during culture is limited, and they usually begin to age after 8-10 generations, so they cannot produce enough cells.

[0004] Therefore, finding a simple, reliable and sufficient cell source is crucial for the application and transformation of MSC. Induced pluripotent stem cells (iPSC) are pluripotent stem cells obtained by inducing adult cells through reprogramming technology and have similar characteristics to embryonic stem cells. iPSC can be cultured and expanded in vitro without restriction, and continuously produce a large number of adult stem cells for application. Therefore, iPSC has gradually become the best cell source for obtaining MSC. At present, the established methods for differentiating various cell types including MSC from iPSC are mostly based on the spontaneous differentiation of embryoid bodies (EBs), which usually takes 30-40 days, is time-consuming, inefficient and uncontrollable. There are also induction methods that first form trophoblast stem cells from pluripotent stem cells, and then further differentiate the trophoblast stem cells into mesenchymal stem cells. This method has problems such as incomplete shedding of upper layer cells, long time consumption, insufficient differentiation of miscellaneous cells, and low purity of mesenchymal stem cells. At the same time, the existing methods for preparing MSCs using iPSC directed induction mostly require adherent culture, digestion, repeated passaging, and involve co-culture of mouse cells, coating of xenobiotic material, flow sorting or viral transfection, etc. Such methods are very complicated and cumbersome to operate, with low yield and unstable quality, and are not suitable for large-scale production. In addition, the culture process introduces exogenous xenobiotics, and their application value is also limited.

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

[0006] The present invention aims to provide a mesenchymal stem cell differentiation induction culture medium and application thereof.

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

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

[0010] In some embodiments of the present invention, the additive further comprises: a volume concentration of 1-10% human platelet lysate, 30-70 mM ascorbic acid, a volume concentration of 0.5%-2% GlutaMax additive, a volume concentration of 0.5%-2% NEAA non-essential amino acids, a volume concentration of 0.5%-2% ITS-X supplement, 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 medium, the amount of the human platelet lysate can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a range consisting of any two of the above values, measured by volume concentration, and is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation 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, 51m 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, is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the GlutaMax additive 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 consisting of any two of the above values, not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the NEAA non-essential amino acid 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 consisting of any two of the above values, not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the ITS-X supplement 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 consisting of any two of the above values, not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of IGF can be 1ng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, or a range consisting of any two of the above values, not limited to the listed values, and other values ​​not listed in the numerical range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of bFGF can be 1ng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, or a range consisting of any two of the above values, not limited to the listed values, and other values ​​not listed in the numerical range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of PDGF-BB can be 1ng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml or a range consisting of any two of the above values, not limited to the listed values, and other unlisted values ​​within the numerical 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 medium is selected from a mesenchymal stem cell serum-free basal medium; the mesenchymal stem cell serum-free basal medium is selected from at least one of high-glucose DMEM, Alpha-MEM, and DMEM / F12 medium.

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

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

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

[0019] Second, replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium for subculture to obtain P3 to P5 generation mesenchymal stem cells or a cell culture comprising P3 to P5 generation mesenchymal stem cells.

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

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

[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 matrix 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% CO 2 , 92-98% humidity.

[0025] In some embodiments of the present invention, in step 2, replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium for subculture comprises 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 with the mesenchymal stem cell induction differentiation medium, and continuing to culture to obtain P1 generation mesenchymal stem cells, and repeating the above subculture steps to obtain P3 to P5 generation mesenchymal stem cells.

[0026] In some embodiments of the present invention, the digestion treatment comprises: removing the supernatant by aspiration, adding calcium- and magnesium-free DPBS to wash the cells, digesting the washed cells, and removing the supernatant by centrifugation to obtain digested cells.

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

[0028] In some embodiments of the present invention, the mesenchymal stem cell expansion medium comprises: a mesenchymal stem cell basal medium and additives, and 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: a volume concentration of 1-10% human platelet lysate, 30-70 mM ascorbic acid, a volume concentration of 0.5%-2% GlutaMax additive, a volume concentration of 0.5%-2% NEAA non-essential amino acids, a volume concentration of 0.5%-2% ITS-X supplement, 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 medium, the amount of the human platelet lysate can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or a range consisting of any two of the above values, measured by volume concentration, and is not limited to the listed values. Other values ​​not listed within the numerical range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation 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 , 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mMl, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM or a range consisting of any two of the above values, is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the GlutaMax additive 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 consisting of any two of the above values, not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the NEAA non-essential amino acid 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 consisting of any two of the above values, not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of the ITS-X supplement 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 consisting of any two of the above values, not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of IGF can be 1ng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, or a range consisting of any two of the above values, not limited to the listed values, and other values ​​not listed in the numerical range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of bFGF can be 1ng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, or a range consisting of any two of the above values, not limited to the listed values, and other values ​​not listed in the numerical range are also applicable. Specifically, in the mesenchymal stem cell induction differentiation medium, the amount of PDGF-BB can be 1ng / ml, 2ng / ml, 3ng / ml, 4ng / ml, 5ng / ml, 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml or a range consisting of any two of the above values, not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

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

[0032] According to the third aspect of the present invention, the present invention also provides a mesenchymal stem cell obtained based on any method according to the second aspect of the present invention.

[0033] In some embodiments of the present invention, the mesenchymal stem cells are multipotent cells that can differentiate into adipocytes, bone cells, chondrocytes, muscle cells, nerve cells and cardiomyocytes.

[0034] In some embodiments of the present invention, the mesenchymal stem cells are capable of expressing CD29, CD73, CD90, CD105, CD166, and CD44 cell surface markers.

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

[0036] In some embodiments of the present invention, the mesenchymal stem cells are mesenchymal stem cells of generation P5 or more, 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 higher than 2%.

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

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

[0039] (2) The present invention significantly improves the differentiation efficiency of mesenchymal stem cells, and even after long-term subculture (e.g., 12 times, or even 15 times or more of subculture), it still shows an excellent effect of stably maintaining the characteristics of mesenchymal stem cells, through which mesenchymal stem cells derived from human pluripotent stem cells can be prepared in large quantities. During the differentiation process, the differentiation pathway is precisely controlled by the combined use of small molecule compounds, achieving stable and efficient differentiation, and finally obtaining mature MSCs. The surface factor expressed by the obtained MSCs is CD90 + 、CD73 + 、CD105 + 、CD14 - 、CD34 - 、CD45 - 、CD19 - and HLA-DR - , adherent growth, and have the ability to differentiate into bone and fat, and no additional flow cytometry screening technology is required to screen the cells.

[0040] (3) Compared with the induction method disclosed in the prior art that pluripotent stem cells first form trophoblast stem cells and then differentiate into mesenchymal stem cells, the above method has the problems of easy shedding of upper layer cells, incomplete and time-consuming, insufficient differentiation of mixed cells, and low purity of mesenchymal stem cells. The induction differentiation medium provided in the present application can be used to directly induce the differentiation of human induced pluripotent stem cells into mesenchymal stem cells. The present application takes a short time and constructs an induction differentiation system with clear components. The serum-free culture system is used to efficiently obtain mesenchymal stem cells with uniform purity and stable performance by directional induction differentiation. Compared with the spontaneous differentiation of embryoid bodies (EBs) disclosed in the prior art, which has the problems of low induction efficiency and easy residual pluripotent cells, time-consuming, labor-intensive, inefficient and uncontrollable, the induction differentiation medium provided in the present application is used to directly induce the differentiation of human induced pluripotent stem cells into mesenchymal stem cells, which simplifies the experimental procedure and has good repeatability. That is, compared with the induction method disclosed in the prior art of first forming an embryoid body (EB) by spontaneous differentiation or first forming trophoblast stem cells by inducing pluripotent stem cells and then differentiating into mesenchymal stem cells, the method provided in the present application takes a short time and efficiently obtains mesenchymal stem cells with uniform purity and stable performance by directional monolayer induction differentiation using a serum-free induction differentiation system with clear components. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The pluripotency identification result of the iPSC cells in Example 1 of the present application;

[0042] Figure 2 The cell morphology of different generations of iMSC derived from iPSC obtained in Example 2 of the present application;

[0043] Figure 3A In the present application example, flow cytometry was used to detect CD90 of iMSCs. + 、CD73 + 、CD105 + 、CD45 - The proportion of

[0044] Figure 3B In the present application example, flow cytometry was used to detect CD14 - 、CD34 - 、CD19 - and the proportion of HLA-DR- phenotype;

[0045] Figure 4 The results of the RT-QPCR test on the expression of MSC-related marker genes OCT4, SNAI2, COL6A2, and TWIST1 in the effect examples of this application;

[0046] Figure 5The results of the test on the expression of iMSC osteogenic specific genes OCN, ALP and RUNX2 in the effect example of the present application;

[0047] Figure 6 The test results of the osteogenic differentiation ability of iMSC in the effect example of this application;

[0048] Figure 7 The results of the test on the expression of the adipogenic specific genes PPARr2 and LPL of iMSC in the effect example of the present application;

[0049] Figure 8 The results of the test on the adipose differentiation ability of iMSC in the effect example of this application;

[0050] Fig. 9 This is a diagram showing the cell culture status of different differentiation induction culture media No. 1-6 in the comparative example of this application;

[0051] Fig.10 This is a diagram showing the cell status of Group A, Group B, and Group C in the comparative example of the present application after differentiation to the 6th day using the iPSC-iMSC induction differentiation medium. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0053] The cells and reagents used in the examples of the present invention are as follows:

[0054] Induced pluripotent stem cells: Product number: hCiPSC-00409, manufacturer: Beijing Beiqi Biopharmaceutical Co., Ltd.;

[0055] Laminin-521 stock solution: Product number: 200-0117, manufacturer: Stemcell;

[0056] DPBS (containing calcium and magnesium): Product number: 14080055, manufacturer: Gibco;

[0057] DPBS (without calcium and magnesium): Product number: C14190500BT, manufacturer: Gibco;

[0058] Vitronectin stock solution: Product number: RP01002, manufacturer: Shouning Biotechnology;

[0059] DMEM / F12: Catalog number: 11330-032, manufacturer: Gibco;

[0060] mTeSRPlus culture medium: Catalog number: 100-0276, manufacturer: Stemcell;

[0061] TrypLE TM Express: Product number: 12604021, Manufacturer: Gibco;

[0062] Y27632: Product number: HY-10071, manufacturer: MCE;

[0063] Versene digestive enzyme: Product number: 15040066 Manufacturer: Gibco;

[0064] Human platelet lysate: Product number: PL-NH-100, manufacturer: Sexton;

[0065] Ascorbic acid: Product number: A8960, manufacturer: Merck;

[0066] GlutaMax Supplement: Product Number: A1286001, Manufacturer: Gibco;

[0067] NEAA non-essential amino acids: Product number: 11140050, manufacturer: Gibco;

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

[0069] IGF: Catalog number: GMP-C023, Manufacturer: Nearshore Protein;

[0070] bFGF: Catalog number: GMP-C046, Manufacturer: Nearshore Protein;

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

[0072] Example 1 Cultivation and subculture of primary cells

[0073] In this embodiment, iPSC cells are used as initial cells. Figure 1 As shown, according to Figure 1 The results shown show that iPSC cells have typical clonal growth characteristics, with clear clone edges, close contact between cells within the clone, high nuclear-cytoplasmic ratio, uniform morphology, and no differentiated cells. The culture and subculture operations of iPSC cells are as follows:

[0074] Laminin-521 coated culture plate: Take the Laminin-521 stock solution and dilute it with DPBS (containing calcium and magnesium) to a final concentration of 5 μg / mL; add the diluted Laminin-521 to the culture plate and place it in a 2-8°C refrigerator overnight or at 37°C 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 use;

[0075] Vitronectin coated culture plate: Take the Vitronectin stock solution and dilute it with DMEM / F12 to a final concentration of 10μg / mL; add the diluted Vitronectin to the culture plate and place it in a 2-8℃ refrigerator 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 use;

[0076] Cell recovery: transfer the thawed iPSC cell suspension to a new 15 mL centrifuge tube, and take the human induced pluripotent stem cell complete medium equilibrated to room temperature and gently add it to the cell suspension, gently shaking the centrifuge tube while adding; transfer the 15 mL centrifuge tube containing the iPSC cell suspension to a low-speed refrigerated centrifuge, centrifuge at room temperature for 3 min under a centrifugal force of 300 g; after centrifugation, discard the supernatant, add human induced pluripotent stem cell complete medium (mTeSR Plus) containing 10 μM Y27632 to resuspend the cells, gently pipette and inoculate them into a preheated 12-well culture plate at a volume ratio of 1:15, and incubate at 37°C, 5% CO 2 The cells were cultured in an incubator with a humidity of 95% and the medium was changed every 20 to 24 hours. Cell passage was started when the cell coverage rate reached more than 80%.

[0077] Cell passaging: When the cell coverage rate reaches more than 80%, take the cell culture plate out of the incubator, wash the cells once with DPBS (without calcium and magnesium), then add Versene digestion enzyme to the culture plate to digest the cells for 3-5 minutes, observe the dissociation state of cell colonies under a microscope, and after the cells are completely dissociated, add human induced pluripotent stem cell complete medium (mTeSRPlus) and gently blow the cell suspension; take out another blank culture plate preheated to 37°C from the incubator, add the cell suspension at a volume ratio of 1:20, and place it at 37°C, 5% CO 2 The cells were cultured overnight in an incubator with a concentration of 50 μg / mL. The cell medium was changed every 20 to 24 hours. When the cell coverage rate reached more than 80%, the next cell passage was started. According to the above method, the cells were passaged to P20, thus obtaining P20 cells.

[0078] Example 2

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

[0080] 1. iPSC culture: The iPSC cells passaged to P20 prepared in Example 1 were cultured normally in a maintenance medium, wherein the maintenance medium used was mTeSR Plus. When the iPSC cells were cultured to a confluence of 80-90%, the iPSC cells were digested into a complete single-cell suspension using Tryple, and resuspended in an iPSC maintenance medium, and Rock inhibitor was added to the iPSC maintenance medium. After the Rock inhibitor was maintained for 24 hours, the medium was replaced with a complete iPSC maintenance medium. The iPSCs were cultured in 12wells, the iPSC maintenance medium used was mTeSR Plus, the Rock inhibitor was Y-27632, the concentration of the Rock inhibitor was 10 μM, and the confluence of the iPSC cells after culture was 30%-50%;

[0081] 2. Induced differentiation of iPSC cells into mesenchymal stem cells (MSC):

[0082] Replace the iPSC maintenance medium in step 1 with mesenchymal stem cell differentiation medium and place in a 5% CO 2 , cultured in a 37°C constant temperature incubator. At this time, the cells were recorded as P0. When the cell confluence reached 80%-90%, the supernatant was removed, and the cells were washed twice with preheated calcium-free and magnesium-free DPBS. Then, preheated Tryple was added to digest the cells until they were in a single cell state. The digestion was terminated, the supernatant was removed after centrifugation, and the cells were resuspended in 1 mL of mesenchymal stem cell induction differentiation medium. The cells were cultured at 5×10 4 cells / cm 2 The cells were inoculated into a well plate pre-coated with Laminin-521 and placed in a 5% CO 2 , cultured in a 37°C constant temperature incubator, at which time the cells are recorded as P1; according to the above steps, the cells are subcultured to P3; wherein the formula of the mesenchymal stem cell induction differentiation medium is: a serum-free basal medium for mesenchymal stem cells (the basal medium is selected from DMEM / F12 medium), containing a 5% volume concentration of human platelet lysate, 50mM ascorbic acid, a 1% volume concentration of GlutaMax additive, a 1% volume concentration of NEAA non-essential amino acids, a 1% volume concentration of ITS-X supplement, 5ng / ml of IGF, 5ng / ml of bFGF, 5ng / ml of PDGF-BB, and 5μM of TGF-β inhibitor (A8301), 5μM of GSK3β inhibitor (CHIR99021), and 8μM of Rock inhibitor (Y-27632);

[0083] 3. iMSC Expansion and Subculture

[0084] When the confluence of the P3 cells obtained in step 2 above reaches about 80%-90%, re-digest the cells until they are in a single cell state, resuspend the cells in 1 mL of mesenchymal stem cell expansion medium, and add 0.9×10 4 cells / cm 2 The cells were inoculated into a well plate pre-coated with Laminin-521 and placed in a 5% CO 2 , cultured in a 37°C constant temperature incubator for 3-5 days, and after the cell growth confluence reached about 80% to 90%, the cells were subcultured in the same manner as above, and subcultured to P12 to obtain P3 to P12 mesenchymal stem cells, respectively; wherein the formula of the mesenchymal stem cell expansion medium is: a serum-free basal medium for mesenchymal stem cells (the basal medium is selected from DMEM / F12 medium), containing a 5% volume concentration of human platelet lysate, 50mM ascorbic acid, a 1% volume concentration of GlutaMax additives, a 1% volume concentration of NEAA non-essential amino acids, a 1% volume concentration of ITS-X supplements, 5ng / ml of IGF, 5ng / ml of bFGF, and 5ng / ml of PDGF-BB.

[0085] Effect Example 1

[0086] The morphology of the mesenchymal stem cells of the P3 generation and subsequent generations prepared in Example 2 was observed. Figure 2 As shown, cells from generations P3 to P12 can all present parallel spiral MSC morphological characteristics, without aging phenomena such as cell volume increase or slowed proliferation, and can stably proliferate and pass on.

[0087] At the same time, the mesenchymal stem cells of the P8 generation prepared in Example 2 were collected for flow cytometry detection. The detection results are shown in FIG3 . + 、CD90 + and CD105 + The expression of CD19 was more than 95%, which was positive. - 、CD14 - 、CD34 - 、CD45 - and HLA-DR - The expression of less than 2% is negative. It should be noted that in this effect experiment, although the present invention only tested the flow cytometry results of P8 cells, according to the latest experimental data, the best can be up to P12.

[0088] Effect Example 2

[0089] In order to verify the effect of iPSCs gradually differentiating into iMSCs in the mesenchymal stem cell induction medium provided by the present invention, the effect example further verifies the above effect by detecting the expression of relevant genes in relevant cells, and the specific steps are as follows:

[0090] The experiment was divided into 3 groups: iMSC group (P1, P3, P5, P7 generations of mesenchymal stem cells prepared in Example 2 of the present invention), iPSC group (prepared in Example 1 of the present invention), ADSC group (adipose-derived mesenchymal stem cells); RNA was extracted from the above 3 groups of cells, and the real-time quantitative RT-QPCR method was used to detect the expression of OCT4, TWIST1, COL6A2 and SNAI2. The specific steps are as follows:

[0091] (1) RNA extraction: RNA was extracted using an RNA purification kit (manufacturer: Quanshijin, 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: Quanshijin, catalog number: AU341-02). The reverse transcription system is as follows:

[0093]

[0094] The above samples were mixed and centrifuged, incubated at 42°C for 15 min, inactivated at 85°C for 5 s, and the obtained cDNA was diluted 2-fold for subsequent reactions;

[0095] (3) Real-time fluorescence quantitative PCR (qRT-PCR): The samples obtained above were subjected to qRT-PCR detection according to the following system. The reaction system is as follows:

[0096]

[0097] The test results obtained by the above method are as follows Figure 4 As shown, according to Figure 4 The results shown in the figure show that the mesenchymal stem cells prepared at different passage times have obvious expressions of TWIST1, COL6A2, and SNAI2. Figure 4 The gene detection results of the P1, P3, P5, and P7 cells shown in Figure 3 show that the cells have clearly transformed from epithelial to mesenchymal (EMT). Combined with the flow cytometry results of the P8 cells shown in Figure 3, it can be further proved that the cells are iMSC cells. OCT4 is a marker gene of iPSC. When iPSCs are successfully differentiated into iMSCs, cells of each generation show that OCT4 is not expressed. Figure 4As shown, cells of generations P1, P3, P5, and P7 hardly expressed OCT4 or expressed it in very small amounts, further indicating that iPSCs can be successfully induced to differentiate into iMSCs by the differentiation induction method provided by the present invention.

[0098] Effect Example 3

[0099] The osteogenic and adipogenic differentiation abilities of the P8 mesenchymal stem cells prepared in Example 2 were further tested, as follows:

[0100] (1) Identification of osteogenic differentiation:

[0101] P8 mesenchymal stem cells were seeded into the cell culture container at an appropriate seeding density, and an appropriate amount of preheated fresh iMSC expansion medium was added. The culture medium was shaken horizontally three times and placed at 37°C and 5% CO. 2 The cells were placed in an incubator with saturated humidity and then shaken three times horizontally in a cross shape for culture. iMSCs spread and grew evenly. When the confluence reached 80%-90%, the culture medium in the container was discarded and replaced with osteogenic differentiation induction medium, which was recorded as day 0. The medium was completely replaced every three days and culture was continued until day 21. The osteogenic differentiation induction medium used was Alpha-MEM medium, which contained 10% FBS, 1% Penicillin-Streptomycin, 10mM β-glycerophosphate (sodium β-glycerophosphate), 10nM Dexamethasone (dexamethasone) and 50μg / ml Ascorbic Acid (L-ascorbic acid).

[0102] Observe and take photos under a light microscope and collect RNA of differentiated cells cultured on day 4, day 14, and day 21 to test the expression of osteogenic-specific genes RUNX2, ALP, and OCN. The results are as follows: Figure 5 As shown. Figure 5 As shown in the results, with the increase of differentiation culture time, the expression of specific genes ALP and OCN gradually increased, and the expression of specific gene RUNX2 first decreased and then increased, indicating that differentiated iMSCs have the ability to differentiate into osteoblasts; after Day 21, the bone-differentiated iMSCs were washed with pure water, and an appropriate volume of Alizarin Red working solution was added, incubated at room temperature in the dark for 20-30 minutes, and then the excess dye was aspirated and discarded, and an appropriate volume of physiological saline or DPBS was added to each well for infiltration, and the wells were observed under a microscope and photographed. The results are shown in Figure 6 As shown, according to Figure 6 As shown in the results, calcium nodules were clearly formed.

[0103] (2) Identification of adipogenic differentiation:

[0104] Inoculate P8 mesenchymal stem cells into the cell culture container at an appropriate seeding density, add an appropriate amount of preheated fresh iMSC expansion medium, shake horizontally three times, and place at 37°C, 5% CO 2 Concentration, in an incubator with saturated humidity, shake horizontally three times again, culture, iMSCs spread and grow evenly, when the culture confluence reaches 80%-90%, the culture medium in the container is discarded, and the adipogenic differentiation induction medium is replaced, which is recorded as day 0. The medium is completely replaced every three days, and the culture is continued until day 21; wherein, the adipogenic differentiation induction medium used is: Alpha-MEM medium, which contains 10% FBS, 1% Penicillin-Streptomycin, 1μM Dexamethasone, 0.5mM IBMX (3-isobutyl-1-methylxanthine), 0.2mM Indomethacin, and 10ug / ml insulin;

[0105] Observe and take photos under a light microscope and collect RNA 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 as follows: Figure 7 As shown. Figure 7 The results shown in the figure show that with the increase of differentiation culture time, the expression level of PPARr2 shows a trend of first increasing and then decreasing, and the expression level of LPL shows a trend of gradually increasing, indicating that differentiated iMSCs have the ability to differentiate into adipocytes. At the same time, during the normal differentiation process, it can be seen that the cells will gradually become wider and shorter, and many round fat particles can be seen in the cells under a high-power microscope. After Day 21, the adipogenically differentiated iMSCs are washed with saline or DPBS, and then washed with 60% isopropanol solution to prevent saline or DPBS from remaining, which will cause the staining solution to precipitate; add an appropriate volume of Oil Red O working solution to the differentiation group and the control group, incubate at room temperature in the dark for 20-60 minutes, then aspirate and discard the excess staining solution, wash with saline or DPBS until no background color is seen, and then add an appropriate volume of saline or DPBS to each well for infiltration, observe under a microscope, and take pictures; the results are as follows Figure 8 As shown, according to Figure 8 The results shown clearly show the formation of lipid droplets.

[0106] Comparative Example

[0107] This comparative example further studies the effects of different differentiation induction media on the properties of mesenchymal stem cells obtained by culturing and differentiating iPSCs, as follows:

[0108] Set up the following groups:

[0109] Medium 1: iMSC expansion medium;

[0110] Medium 2: iMSC expansion medium supplemented with 5 μM GSK3β inhibitor (CHIR99021);

[0111] Medium 3: iMSC expansion medium supplemented with 5 μM TGF-β inhibitor (A8301);

[0112] Medium 4: iMSC expansion medium supplemented with 8 μM Rock inhibitor (Y-27632);

[0113] Medium 5: iMSC expansion medium supplemented with 5 μM GSK3β inhibitor (CHIR99021) and 5 μM TGF-β inhibitor (A8301);

[0114] Medium 6: iMSC expansion medium supplemented with 5 μM GSK3β inhibitor (CHIR99021), 5 μM TGF-β inhibitor (A8301), and 8 μM Rock inhibitor (Y-27632);

[0115] Among them, the iMSC expansion medium formula is: mesenchymal stem cell serum-free basal medium (basal medium is 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, 5ng / ml PDGF-BB;

[0116] After the iPSCs prepared in Example 1 were passaged, the old medium was removed and the cells were divided into 8 portions. After replacing the above 8 culture media, the cells were placed in a 5% CO 2、 The cells were cultured in a 37°C constant temperature incubator, and then the corresponding fresh differentiation medium was replaced every day. The cell morphology and proliferation were observed. The cells were passaged when the confluence reached 85% to 90%. The cell morphology was observed during the differentiation of iPSC to iMSC. Fig. 9 As shown, according to Fig. 9 The results shown show that the cells in medium 1 had basically no changes in morphology and basically no cell proliferation; the cells in medium 2 gradually died during differentiation; the cells in medium 3 had basically no changes in morphology; no obvious proliferation was observed when cells in medium 4 and 5 were passaged to P1; the cells in medium 6 were passaged to P2, and normal adhesion was observed, with most cells showing short spindle shape.

[0117] The effects of inducing differentiation culture of iPSCs by the following three groups of induction differentiation medium on the preparation of mesenchymal stem cells were further compared as follows:

[0118] Group A culture medium: medium 6;

[0119] 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;

[0120] Culture medium for group C: basal culture 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;

[0121] The iPSC single cells prepared in Example 1 were inoculated 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 was replaced in group A, group B, and group C respectively. The corresponding fresh induction differentiation medium was replaced every day. The culture was continued until the 6th day, and the corresponding cell morphology was observed. The results are as follows: Fig.10 As shown, according to Fig.10 The results shown show that the cells corresponding to the No. 6 culture medium in group A were in a good state of differentiation, with mesenchymal-like cells observed under the microscope, while cells in groups B and C died to varying degrees after being cultured for 6 days.

[0122] comprehensive Fig. 9 , Fig.10 The cell morphology and proliferation results further indicate that mesenchymal stem cells with excellent performance in various aspects can be obtained by directly differentiating and culturing iPSCs using the differentiation induction medium provided by the present invention.

[0123] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of the present invention are within the scope of protection of the present invention.

Claims

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

2. The mesenchymal stem cell differentiation induction 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, PDGF-BB; Preferably, the additive further comprises: human platelet lysate with a volume concentration of 1-10%, 30-70 mM ascorbic acid, GlutaMax additive with a volume concentration of 0.5%-2%, NEAA non-essential amino acids with a volume concentration of 0.5%-2%, ITS-X supplement with 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 differentiation induction 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 differentiation induction medium according to claim 1, characterized in that The mesenchymal stem cell basal culture medium is selected from a mesenchymal stem cell serum-free basal culture medium; the mesenchymal stem cell serum-free 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 the differentiation of human induced pluripotent stem cells / embryonic stem cells into mesenchymal stem cells, characterized in that: The method comprises: selecting the mesenchymal stem cell induction differentiation medium as described in any one of claims 1 to 4 to perform subculture on human induced pluripotent stem cells and / or embryonic stem cells, and directly inducing differentiation of the human induced pluripotent stem cells and / or embryonic stem cells into mesenchymal stem cells.

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

1. Providing cell cultures of human pluripotent stem cells or embryonic stem cells; Second, replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium for subculture to obtain P3 to P5 generation mesenchymal stem cells or a cell culture comprising P3 to P5 generation mesenchymal stem cells.

7. The method according to claim 6, characterized in that In step 1, the culturing step of the cell culture comprises: using a culture medium without xenogeneic animal components to culture the human pluripotent stem cells or embryonic stem cells in a culture method without feeder cells; preferably, the culturing step comprises first culturing the human pluripotent stem cells or embryonic stem cells normally in a maintenance culture medium, culturing the cells to a confluence of 80-90%, digesting the iPSC cells into a complete single cell suspension using Tryple, resuspending the cells in an iPSC maintenance culture medium, and adding Rock inhibitor to the iPSC maintenance culture medium, and replacing the culture medium with a complete iPSC maintenance culture medium after the Rock inhibitor is maintained for 24 hours to obtain the cell culture with a confluence of 30% to 50%; Preferably, the maintenance 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 matrix gel used in the culture process is selected from at least one of Laminin-521, Vitronectin, and Matrigel; Preferably, the culture conditions of the culture process include: culturing in a constant temperature incubator at 35-40° C., 4-6% CO 2 , and 92-98% humidity.

8. The method according to claim 6, characterized in that In step 2, replacing the culture medium in the cell culture with the mesenchymal stem cell induction differentiation medium for subculture comprises 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% as P0 generation, digesting the cells, resuspending the digested cells with the mesenchymal stem cell induction differentiation medium, continuing to culture, obtaining P1 generation mesenchymal stem cells, repeating the above subculture steps, obtaining P3 to P5 generation mesenchymal stem cells; Preferably, the digestion treatment comprises: removing the supernatant by suction, adding calcium- and magnesium-free DPBS to wash the cells, digesting the washed cells, and removing the supernatant by centrifugation to obtain digested cells.

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

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

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