Mesenchymal stem cells derived from definitive endoderm cells and differentiation method and application thereof

Abstract

The present invention discloses a mesenchymal stem cell derived from a definitive endoderm cell, a differentiation method and an application thereof, and relates to the technical field of mesenchymal stem cells. The differentiation method comprises: inducing human pluripotent stem cells to differentiate into mesendoderm cells by culturing in a first differentiation medium containing a WNT pathway activator and Activin A for 1-2 days; inducing the formation of definitive endoderm cells (DE) by culturing in a second differentiation medium containing no WNT pathway activator and Activin A for 2-8 days; inducing the acquisition of midgut, hindgut and foregut cell fate by culturing in a MSC generation medium for 2-4 days; inducing the generation of a cell population containing mesenchymal stem cells by culturing in a MSC generation medium for 8-10 days; performing digestion and passage 2-3 times to obtain mesenchymal stem cells (DE-MSCs) derived from definitive endoderm cells. It can secrete anti-inflammatory factors under in vitro stimulation and has the potential to treat colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cells, and in particular to a mesenchymal stem cell derived from a definitive endoderm cell, and a differentiation method and application thereof. Background Art

[0002] At present, traditional therapies based on chemical drugs, biological drugs and surgical treatments still cannot solve many disease problems, while regenerative medicine based on stem cell technology is gradually being applied in clinic and showing great potential. Compared with other types of stem cells, mesenchymal stem cells (MSCs) have the characteristics of low immunogenicity (i.e., the major histocompatibility complex MHC I is lowly expressed in MSCs, and MHC II is not expressed in MSCs), so MSCs are more suitable for allogeneic applications and have become an ideal candidate for cell therapy.

[0003] Mesenchymal stem cells (MSCs) have multiple therapeutic potentials for tissue engineering and regenerative medicine. They can not only directly differentiate into tissue-specific cells to repair wounds but also stimulate tissue repair by releasing paracrine forms of anti-inflammatory cytokines, anti-apoptotic factors, and growth factors, which can be used to treat a variety of diseases, including inflammatory diseases and autoimmune diseases.

[0004] In addition, existing literature records that MSCs can treat respiratory diseases, digestive tract diseases, liver diseases, pancreatitis, colorectal inflammation and other diseases.

[0005] Although MSCs have achieved positive results in the treatment of respiratory and digestive tract diseases, and many clinical trials have proven that MSCs therapy is safe and effective, there are still great differences in the results and conclusions of these studies. The most reasonable explanation is: 1) The source of MSCs donors is too broad. Traditionally, mesenchymal stem cells are derived from human tissues, mainly bone marrow and fat. MSCs can also be isolated from umbilical cord blood, placenta, amnion, dental pulp and other tissues. From the perspective of embryonic development, bone marrow, fat, dental pulp, umbilical cord, placenta, amnion, etc. are derived from mesoderm, ectoderm or trophoblast, and the use of mesoderm, ectoderm or trophoblast-derived MSCs is not necessarily suitable for endoderm (progenitor cells of respiratory and digestive organs)-related diseases; 2) The culture schemes of MSCs from different donor sources also vary greatly. Therefore, it is crucial to produce homogeneous and germ layer-matched MSCs for treatment.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a mesenchymal stem cell derived from a definitive endoderm cell and a differentiation method and application thereof.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a method for differentiating mesenchymal stem cells derived from definitive endoderm cells, comprising:

[0010] The first differentiation stage: Inducing human pluripotent stem cells to differentiate into mesendoderm cells by culturing in the first differentiation medium containing WNT pathway activators and Activin A for 1-2 days;

[0011] The second differentiation stage: culture in the second differentiation medium without WNT pathway activator and containing Activin A for 2-8 days to induce the formation of definitive endoderm cells (DE);

[0012] The third differentiation stage: culturing in MSC generation medium for 2-4 days to induce the definitive endoderm cells to acquire the fate of midgut cells, hindgut cells and foregut cells;

[0013] Fourth differentiation stage: continue to culture in MSC generation medium for 8-14 days to induce the generation of cell populations containing mesenchymal stem cells;

[0014] Fifth differentiation stage: Digestion and passage are performed 2-3 times to obtain definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs).

[0015] In a second aspect, the present invention provides a definitive endoderm cell-derived mesenchymal stem cell, which is differentiated by the method for generating definitive endoderm cell-derived mesenchymal stem cells from human pluripotent stem cells as described in any one of the aforementioned embodiments.

[0016] In a third aspect, the present invention provides use of mesenchymal stem cells derived from definitive endoderm cells as described in the aforementioned embodiments in the preparation of a drug for treating intestinal-related diseases.

[0017] In a fourth aspect, the present invention provides a mesenchymal stem cell differentiated cell, which is differentiated in vitro from the mesenchymal stem cells derived from the definitive endoderm cells as described in the above embodiments;

[0018] Preferably, the cells differentiated from the mesenchymal stem cells include at least one of adipocytes, chondrocytes and osteoblasts.

[0019] In a fifth aspect, the present invention provides a kit for inducing human pluripotent stem cells to generate definitive endoderm cell-derived mesenchymal stem cells, which includes reagents for implementing the differentiation method of definitive endoderm cell-derived mesenchymal stem cells as described in the above embodiments.

[0020] The present invention has the following beneficial effects:

[0021] The differentiation method of mesenchymal stem cells derived from definitive endoderm cells provided in the present application uses a culture medium with clear components to induce hPSC to differentiate into definitive endoderm cells (DE) and mesenchymal stem cells (DE-MSCs) derived specifically from definitive endoderm cells. More importantly, these cells can acquire the fate of midgut, hindgut and foregut cells through different induction methods. DE-MSCs obtained through the foregut cell fate pathway are similar to MSCs derived from human lungs, liver and pancreas, and are suitable for treating body lung, liver and pancreas-related diseases represented by COVID-19, cirrhosis, pancreatitis, etc.; while DE-MSCs obtained through the midgut / hindgut cell fate pathway are similar to MSCs derived from human small intestine and large intestine, and are suitable for treating intestinal-related diseases represented by colitis and Crohn's disease. The present application provides an in vitro differentiation method for efficiently preparing mesenchymal stem cells derived from definitive endoderm derived from human pluripotent stem cells. And these DE-MSCs can secrete anti-inflammatory factors under in vitro stimulation conditions, and further, these DE-MSCs injected intraperitoneally have the ability to treat colitis mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The differentiation protocol of definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) provided in Example 1;

[0024] Figure 2 The figure shows the changes in cell morphology (upper figure) and the changes in the expression of the definitive endoderm cell marker SOX17 (lower figure) over time from day 0 to day 24 in Example 1;

[0025] Figure 3 For Example 1 SOX17 mRNA and mesenchymal stem cell characteristic genes CD44 , CD73 , CD105 Changes in mRNA expression levels over time;

[0026] Figure 4 The percentage of cells positive for mesenchymal stem cell markers CD44, CD73, CD105, and CD45 on day 24 in Example 1;

[0027] Figure 5The percentage of cells positive for the definitive endoderm cell marker SOX17 and the percentage of cells positive for the mesenchymal stem cell markers CD44, CD73 and CD105 in Example 1;

[0028] Figure 6 The total number of cells of the definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) in Example 1 changes over time;

[0029] Figure 7 The mesenchymal stem cells (DE-MSCs) derived from the definitive endoderm cells in Example 1 are further differentiated into adipocytes (which can be stained red by Oil Red O dye), chondrocytes (which can be stained blue by Alcian blue dye), and osteocytes (which can be stained black by Von Kossa dye);

[0030] Figure 8 A schematic diagram of the differentiation scheme of definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) provided in Example 2;

[0031] Fig. 9 The SOX17-positive DE cells purified in Example 2 (only the top 48% of DE cells with the highest expression intensity were selected, and the purified cells were returned to the MSC generation conditions to induce them to further form DE-MSCs);

[0032] Fig.10 The cell morphology on the 15th and 24th days in Example 2;

[0033] Fig.11 is the percentage of CD44, CD73, and CD105 positive cells on day 24 in Example 2;

[0034] Fig.12 A schematic diagram of the differentiation scheme of definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) provided in Example 3;

[0035] Fig.13 The RT-qPCR analysis in Example 3 was used to test the effects of CHIR99021, SB431542, XAV939 and their combination on the 15th day. CD44 , CD73 , CD105 Effects of mRNA expression levels;

[0036] Fig.14 The cell morphology of DE-MSCs (FBS), DE-MSCs (CHIR), and DE-MSCs (CHIR / SB) on day 24 in Example 3;

[0037] Fig.15is the percentage of CD44, CD73, CD105, PDGFRβ, and CD45 positive cells of DE-MSCs (FBS), DE-MSCs (CHIR), and DE-MSCs (CHIR / SB) on day 24 in Example 3;

[0038] Fig.16 The cell morphology of mesenchymal stem cells (DE-MSCs) derived from human induced pluripotent stem cells NL-1 and human embryonic stem cells H1 in Example 4 on day 24;

[0039] Fig.17 The percentage of CD44 and CD105 positive cells of mesenchymal stem cells (DE-MSCs) derived from human induced pluripotent stem cells NL-1 and human embryonic stem cells H1 on day 24 in Example 4;

[0040] Fig.18 The weight changes of the healthy mice, the DSS+PBS mice, the DSS+PBS+DE-MSCs (FBS) mice, the DSS+PBS+DE-MSCs (CHIR) mice, the DSS+PBS+DE-MSCs (CHIR / SB) mice, and the DSS+PBS+UC-MSCs mice in Example 5 from day 0 to day 14;

[0041] Fig.19 The colon lengths of the healthy mice, the DSS+PBS mice, the DSS+PBS+DE-MSCs (FBS) mice, the DSS+PBS+DE-MSCs (CHIR) mice, the DSS+PBS+DE-MSCs (CHIR / SB) mice, and the DSS+PBS+UC-MSCs mice in Example 5 on the 14th day;

[0042] Fig. 20 These are the colon images of the healthy mice, DSS+PBS mice, DSS+PBS+DE-MSCs (FBS) mice, DSS+PBS+DE-MSCs (CHIR) mice, DSS+PBS+DE-MSCs (CHIR / SB) mice, and DSS+PBS+UC-MSCs mice in Example 5 on the 14th day. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0044] The present invention provides a method for differentiating mesenchymal stem cells derived from definitive endoderm cells, which comprises: inducing the differentiation of human pluripotent stem cells into definitive endoderm cells (DE), inducing the differentiation of definitive endoderm cells into mesenchymal stem cells (MSCs), and the mesenchymal stem cells derived from definitive endoderm cells via human pluripotent stem cells are collectively referred to as DE-MSCs.

[0045] Specifically, the differentiation method provided in this application comprises the following steps:

[0046] S1. Cultivate and passage human pluripotent stem cells, and start differentiation when the cells grow to 30-60%.

[0047] Human pluripotent stem cells were cultured in E8 medium, and fresh E8 medium was replaced every day. When the cell density reached 70-80%, the cells were passaged. When passaged, human pluripotent stem cells were washed 2-3 times with DPBS-EDTA, then incubated at room temperature for 4-10 minutes, aspirated DPBS-EDTA, and added with stem maintenance medium containing 5 μM ROCK inhibitor. After resuspending the cells, they were passaged at a density of 1:5-1:7 and added to the well plate pre-coated with Matrigel. After the cells adhered for 1 day, fresh E8 medium was replaced and continued to be cultured. When the cells grew to 30-60%, they began to differentiate.

[0048] The components of E8 culture medium include: DMEM / F12 (basal culture medium, purchased from ThermoFisher, catalog number 11330-032), L-ascorbic acid-2-phosphate magnesium 60-70 mg / L, sodium selenite 10-15 μg / L, transferrin 8-12 μg / ml, insulin 8-12 μg / ml, FGF2 90-110 ng / ml, TGFβ 1-2 ng / ml.

[0049] Human pluripotent stem cells include at least one of human embryonic stem cells and human induced pluripotent stem cells; the human embryonic stem cells are stem cells separated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development; preferably, human embryonic stem cells include human embryonic stem cells H1 or human embryonic stem cells H9; human induced pluripotent stem cells include human induced pluripotent stem cells NL-1.

[0050] The human embryonic stem cells H1 and H9 in this application were purchased from WiCell Research Institute, Inc. (Madison, Wisconsin, USA, http: / / www.wicell.org), and the human induced pluripotent stem cell NL-1 was a cell line preserved in this laboratory (hPSCreg number CRMi003-A).

[0051] S2, differentiation stage.

[0052] (1) The first differentiation stage.

[0053] The human pluripotent stem cells are induced to differentiate into mesendoderm by culturing in a first differentiation medium containing a WNT pathway activator and Activin A for 1-2 days.

[0054] The WNT pathway activator includes at least one of CHIR99021, CHIR99021-HCl, Wnt3a ligand, Wnt5a ligand and BIO; the treatment concentration of CHIR99021 or CHIR99021-HCl is 1-5 μM.

[0055] Activins Activin A is a secreted protein of the transforming growth factor (TGF) β family that controls the development of the embryonic axis into functional foregut-derived tissue and regulates the growth and differentiation of various cells. It is also widely used in stem cell research to induce embryonic stem cells to differentiate into endoderm cells.

[0056] In the present application, the combined use of WNT pathway activator and Activin A can exert a more significant inducing effect on mesendoderm cell differentiation.

[0057] The basic components of the "first differentiation medium" herein are the same as the components of the conventional culture medium for inducing stem cell differentiation. "Using the first differentiation medium containing WNT pathway activator and Activin A" can be understood as adding WNT pathway activator and Activin A to the basic components of the existing first differentiation medium. That is, in this application, the first differentiation medium is obtained by adding WNT pathway activator and Activin A to the basic components. The added amount of WNT pathway activator is 1-5 μM, and the added amount of Activin A is 10-100 ng / ml.

[0058] Specifically, the basic components include DMEM / F12, L-ascorbic acid-2-magnesium phosphate 60-70 mg / L, sodium selenite 10-15 μg / L, transferrin 8-12 μg / ml, Chemical defined lipid concentrate 1× and penicillin-streptomycin 1%.

[0059] Culture for 1-2 days, that is, the culture time is calculated from the 0th day of differentiation. "Day 0" in this article refers to the starting point of inducing differentiation, that is, 0-24 hours. Culture for 1-2 days can be understood as culture at any time within 0-48 hours after the start of differentiation using the first differentiation medium containing WNT pathway activator and Activin A. Preferably, the differentiation culture time is 1 day.

[0060] Specifically, in the present application, the time when the WNT pathway activator and Activin A are contained in the differentiation medium can be any time between 1 and 2 days, specifically any time point or any time period between 0 days, 0.5 days, 1 day, 1.5 days, and 2 days. It should be noted that when the time for culturing with a differentiation medium containing a specific agent is a time period, the culture medium can be selectively replaced several times in the time period so that the differentiation medium contains a specific agent. For example, in a time period of 1 to 2 days, a new culture medium can be replaced every day, every 10 hours, or every 20 hours to make the process of inducing differentiation more stable and effective. Subsequent similar situations are understood in the same way and will not be repeated.

[0061] (2) The second differentiation stage.

[0062] The cells are cultured in a second differentiation medium containing Activin A but without WNT pathway activators for 2-8 days to induce the formation of definitive endoderm cells.

[0063] In the second differentiation stage, the present application removes the WNT pathway activator and only adds Activin A to the basic components as the second differentiation medium for culture. At this time, the formation of definitive endoderm cells (DE) can be further induced. Generally, high-purity definitive endoderm cells (DE) can be obtained by treating with Activin A (10-100 ng / ml) for two days, but extending the treatment time of Activin A (10-100 ng / ml) to 4 days, 6 days and 8 days can also obtain high-purity definitive endoderm cells (DE), and DE-MSCs can still be obtained according to the subsequent steps. Therefore, the second differentiation stage in the present application can be cultured for 2-8 days, preferably 2 days.

[0064] Specifically, the basic components include DMEM / F12 (basal culture medium, purchased from ThermoFisher, catalog number 11330-032), L-ascorbic acid-2-phosphate magnesium (effective concentration 60-70 mg / L), sodium selenite (effective concentration 10-15 μg / L), transferrin (effective concentration 8-12 μg / ml), Chemical defined lipid concentrate (chemically defined lipid concentrate, purchased from ThermoFisher, catalog number 11905031) 1× and penicillin-streptomycin (effective concentration 1%).

[0065] Preferably, after differentiation to obtain definitive endoderm cells (DE), flow cytometry sorting technology is further used to purify the definitive endoderm cells (DE). Purifying DE includes: harvesting cells with cell digestion solution, neutralizing with cell neutralization solution, washing and resuspending the cells, and purifying them with flow cytometry.

[0066] (3) The third differentiation stage.

[0067] The definitive endoderm cells are cultured in MSC generation medium for 2-4 days to induce the fate of midgut cells, hindgut cells and foregut cells, wherein midgut cells are characterized by expressing the biomarker PDX1, hindgut cells are characterized by expressing the biomarker CDX2, and foregut cells are characterized by expressing the biomarker SOX2.

[0068] A cell signaling pathway regulator may be added to the MSC generation medium in the third differentiation stage. In the present application, the cell signaling pathway regulator includes at least one of a WNT pathway activator, a WNT pathway inhibitor, and a TGF pathway inhibitor.

[0069] Among them, the WNT pathway activator includes at least one of CHIR99021, CHIR99021-HCl, Wnt3a ligand, Wnt5a ligand and BIO; the treatment concentration of the WNT pathway activator is 1-5 μM; the WNT pathway inhibitor includes at least one of XAV939, IWP2 and IWR-1, and the treatment concentration of the WNT pathway inhibitor is 1-10 μM; the TGF pathway inhibitor includes one of SB431542, RepSox, SD-208, GW788388, A-77-01 and A-83-01; the treatment concentration of the TGF pathway inhibitor is 1-10 μM.

[0070] The composition of MSC generation medium when serum-free includes αMEM, L-ascorbic acid-2-phosphate magnesium 60-70 mg / L, sodium selenite 10-15 μg / L, insulin 8-12 μg / ml, FGF2 90-110 ng / ml, B-27™ Supplement 1×, GlutaMAX™ Supplement1×, NEAA 1×, and penicillin-streptomycin 1%; alternatively, the composition of MSC generation medium when serum-supplemented includes αMEM, FBS 10%-20%, L-ascorbic acid-2-phosphate magnesium 60-70 mg / L, sodium selenite 10-15 μg / L, insulin 8-12 μg / ml, FGF2 90-110 ng / ml, B-27™ Supplement 1×, GlutaMAX™ Supplement1×, NEAA 1×, and penicillin-streptomycin 1%.

[0071] (4) Fourth differentiation stage

[0072] Continue to culture in the MSC generation medium for 8-10 days to induce the generation of a cell population containing mesenchymal stem cells; in the fourth differentiation stage, only use the MSC generation medium to culture the cells without adding cell signaling pathway regulators, and change the medium every two days to induce the generation of a cell population containing mesenchymal stem cells.

[0073] (5) Fifth differentiation stage

[0074] Conventional digestion and subculturing were performed using trypsin digestion solution. After 2-3 subculturings, definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) were obtained.

[0075] The gene expression profile of the definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) obtained by the above differentiation method is similar to the gene expression profile of the endoderm organs; and these DE-MSCs are able to secrete anti-inflammatory factors under in vitro stimulation conditions. Furthermore, these DE-MSCs injected intraperitoneally have the potential to treat colitis in mice and can be widely used in the preparation of drugs for the treatment of intestinal-related diseases, specifically, intestinal-related diseases include but are not limited to colitis, respiratory diseases and digestive tract inflammatory diseases.

[0076] Correspondingly, the present application also provides a kit for inducing human pluripotent stem cells to generate mesenchymal stem cells derived from definitive endoderm cells, which includes reagents for implementing the above-mentioned differentiation method of mesenchymal stem cells derived from definitive endoderm cells.

[0077] In addition, the present application also provides a mesenchymal stem cell differentiation cell, which is differentiated in vitro from the mesenchymal stem cells derived from the above-mentioned definitive endoderm cells; the mesenchymal stem cell differentiation cell includes at least one of adipose cells, chondrocytes and osteoblasts.

[0078] The materials and reagents used in the following examples are derived from:

[0079] CHIR99021 (CT99021) HCl, full name WNT pathway activator, purchased from Selleckchem, catalog number S2924;

[0080] Activin A, full name: a member of the transforming growth factor superfamily, commercially available from R&D Systems, catalog number 338-AC-500 / CF;

[0081] TGFβ, full name transforming growth factor β, purchased from R&D Systems, catalog number 240-B-500;

[0082] FGF2, full name fibroblast growth factor 2, is commercially available from Peprotech, catalog number 100-18C:

[0083] XAV939, full name WNT pathway inhibitor, commercially available from Selleckchem, catalog number S1180;

[0084] SB432542, full name TGF pathway inhibitor, purchased from Selleckchem, catalog number S1067;

[0085] DMEM / F12 basal medium, purchased from ThermoFisher, catalog number 11330-032;

[0086] Chemical defined lipid concentrate is a chemically defined lipid concentrate purchased from ThermoFisher, catalog number 11905031;

[0087] αMEM is the basal medium, purchased from ThermoFisher, catalog number 12571063;

[0088] FBS, full name fetal bovine serum, purchased from Gibco, catalog number 10437028;

[0089] B-27™ Supplement, full name B-27™ Supplement, commercially available from ThermoFisher, catalog number 17504044, with an initial concentration of 50×;

[0090] GlutaMAX™ Supplement, full name GlutaMAX™ Supplement, commercially available from Gibco, catalog number 35050061, with an initial concentration of 100×;

[0091] MEM Non-Essential Amino Acids Solution (NEAA), full name MEM non-essential amino acid solution, purchased from Gibco, catalog number 11140050, with an initial concentration of 100×;

[0092] DPBS-EDTA, purchased from Cell system, catalog number 4Z0-610;

[0093] Human embryonic stem cells H1: purchased from WiCell Research Institute, Inc. (Madison, Wisconsin, USA, http: / / www.wicell.org);

[0094] Human embryonic stem cells H9: purchased from WiCell Research Institute, Inc. (Madison, Wisconsin, USA, http: / / www.wicell.org);

[0095] Human induced pluripotent stem cell NL-1: a cell line preserved in our laboratory, with the number CRMi003-A in the hPSCreg human induced pluripotent stem cell registry (https: / / hpscreg.eu).

[0096] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0097] Example 1

[0098] This embodiment provides a method for differentiating definitive endoderm (DE)-derived mesenchymal stem cells (DE-MSCs), which specifically adopts a method of gradually differentiating stem cells in vitro.

[0099] The specific implementation is as follows: Human embryonic stem cell H9 (SOX17-GFP reporter cell line, SOX17 is a specific biomarker for definitive endoderm cells, and the SOX17-GFP reporter cell line will show green fluorescence when differentiating into definitive endoderm cells. Therefore, the formation of definitive endoderm cells can be observed by fluorescence microscopy or flow cytometry) is cultured in E8 medium, and fresh medium is replaced every day. When the cell density reaches 70-80%, it is passaged. First, wash with DPBS-EDTA twice, then incubate at room temperature for 5 minutes, aspirate DPBS-EDTA, and add E8 medium containing ROCK inhibitor 5 μM. After resuspending the cells, pass them at a density of 1:6 and add them to the well plate pre-coated with Matrigel. After the cells adhere to the wall for 1 day, change E8 medium and continue to culture. When the cells grow to 30-60%, differentiation begins. On the first day, the first differentiation medium with CHIR99021-HCl 5 μM and Activin A 100 ng / ml added to the basic components is used for culture. On the second and third days of differentiation, the cells were cultured in a second differentiation medium containing basic ingredients supplemented with 100 ng / ml of Activin A to induce the formation of definitive endoderm cells. On the third to fifth days of differentiation, the medium was replaced with fresh MSC generation medium; on the fifth to fifteenth days of differentiation, the medium was replaced with fresh MSC generation medium, and the medium was changed every two days to induce the generation of a cell population containing mesenchymal stem cells; after the fifteenth day, the cells were passaged using a trypsin-based digestion solution. Generally, after 2-3 passages, definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) that meet the mesenchymal stem cell standards (ISCT standards, 2006) can be obtained.

[0100] The components of E8 culture medium include: DMEM / F12 (basal culture medium, purchased from ThermoFisher, catalog number: 11330-032), L-ascorbic acid-2-phosphate magnesium 64 mg / L, sodium selenite 13.6 μg / L, transferrin 10 μg / ml, insulin 10 μg / ml, FGF2 100 ng / ml, TGFβ 1.74 ng / ml.

[0101] The basic components of the first differentiation medium and the second differentiation medium include: DMEM / F12, L-ascorbic acid-2-phosphate magnesium 64 mg / L, sodium selenite 13.6 μg / L, transferrin 10 μg / ml, Chemical defined lipid concentrate 1× and penicillin-streptomycin 1%.

[0102] The components of MSC generation medium (with serum) include: αMEM, FBS 10-20%, L-ascorbic acid-2-phosphate magnesium 64 mg / L, sodium selenite 13.6 μg / L, transferrin 10 μg / ml, insulin 10 μg / ml, FGF2 100 ng / ml, B-27™ Supplement 1×, GlutaMAX™ Supplement 1×, MEM Non-Essential Amino Acids Solution 1×, and penicillin-streptomycin 1%.

[0103] The components of MSC generation medium (serum-free) include: αMEM, L-ascorbic acid-2-phosphate magnesium 64 mg / L, sodium selenite 13.6 μg / L, transferrin 10 μg / ml, insulin 10 μg / ml, FGF2 100 ng / ml, B-27™ Supplement 1×, GlutaMAX™ Supplement 1×, MEM Non-Essential Amino Acids Solution 1×, and penicillin-streptomycin 1%.

[0104] The mesenchymal stem cells derived from the definitive endoderm cells prepared in Example 1 were tested, and the testing method was as follows:

[0105] qPCR was measured using a fluorescent quantitative PCR detector (commercially available from ThermoFisher, model QuantStudio7), total mRNA was extracted using RNAiso-plus (commercially available from TAKARA, product number 108-95-2), and reverse transcription from mRNA to cDNA was performed using a high-capacity cDNA reverse transcription kit (commercially available from Applied Biosystems, product number 4368813). Real-time quantitative PCR was performed using SYBR Premix Ex Taq (commercially available from TAKARA, product number RR420) and the Quantstudio-7 system. The relative amount of amplified nucleotide fragments was calculated by the 2^(-ΔCt) method. The expression level was normalized to the housekeeping gene TBP and compared with undifferentiated human embryonic stem cells.

[0106] Immunofluorescence was measured using an immunofluorescence analyzer (commercially purchased from ThermoFisher, model EVOS Cell ImagingSystem FL). The cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, rinsed with 1× PBS (5 minutes each, 3 times), permeabilized with 0.5% Triton X-100 for 20 minutes, and then rinsed with 1× PBS (5 minutes each, 3 times). The nuclei were stained with Hoechst 33342 (commercially purchased from Abnova, catalog number U0334) for 20 minutes at room temperature. The cells were then rinsed with 1× PBS (5 minutes each, 3 times), incubated with antibodies at 4°C overnight, rinsed with 1× PBS, incubated with fluorescent secondary antibodies for 2 hours, and finally rinsed with 1× PBS for imaging.

[0107] Flow cytometry was measured using a flow cytometer (commercially available from BD, model BD C6), and cells were harvested by TrypLE (37°C, 5 minutes) and neutralized with 5% FBS. After washing with DPBS with 1% BSA, the antibodies were incubated, and after washing, the cells were resuspended in DPBS and analyzed by BD C6.

[0108] Please refer to the differentiation scheme provided in this example Figure 1 For test results, please refer to Figure 2-Figure 7 .

[0109] from Figure 2 It can be seen that on days 3 and 5, the definitive endoderm-specific protein SOX17 is highly expressed, indicating that after the first and second stages of differentiation, high-purity definitive endoderm cells have been obtained. On day 24, spindle cells can be observed, and their SOX17 is negative, indicating that these definitive endoderm cells have left their original cell fate and differentiated into cells with mesenchymal stem cell morphology (spindle).

[0110] from Figure 3 The cells can be cultured on days 3, 5, 10, 15, 18 (1st passage, P1), 21 (2nd passage, P2), and 24 (3rd passage, P3). SOX17 The gradual decrease in mRNA expression levels and the characteristic genes of mesenchymal stem cells CD44, CD73、CD105 The gradual increase in mRNA expression levels (three repeated experiments, *p<0.05) showed that the definitive endoderm cells had left their original cell fate and differentiated into cells with mesenchymal stem cell gene characteristics ( CD44, CD73、CD105 ) cells.

[0111] from Figure 4It can be seen that mesenchymal stem cells derived from definitive endoderm cells can highly express the positive biomarkers characteristic of mesenchymal stem cells (CD44, CD73, CD105), while not expressing the negative biomarkers characteristic of mesenchymal stem cells (CD45) (three repeated experiments, *p<0.05).

[0112] from Figure 5 It can be seen that the percentage of SOX17-positive cells gradually decreased on days 0, 3, 5, 10, 15, 18 (P1), 21 (P2), and 24 (P3), while the percentage of CD44, CD73, and CD105-positive cells gradually increased on days 18 (P1), 21 (P2), and 24 (P3) (three repeated experiments, *p<0.05).

[0113] Figure 6 Mesenchymal stem cells generated in vitro from definitive endoderm cells have the ability to proliferate in vitro.

[0114] Figure 7 Mesenchymal stem cells generated in vitro from definitive endoderm cells have the ability to continue to differentiate into adipocytes, chondrocytes, and osteoblasts in vitro.

[0115] comprehensive Figure 2-Figure 7 It can be seen that in the first and second stages of differentiation, high-purity definitive endoderm cells can be obtained by the method of this embodiment. After the third, fourth, and fifth stages, mesenchymal stem cells generated from definitive endoderm cells can be obtained by the method of this embodiment, and these mesenchymal stem cells meet the requirements of the International Stem Cell Association (ISCT Standard, 2006) for mesenchymal stem cell standards.

[0116] Example 2

[0117] This embodiment provides a method for differentiating mesenchymal stem cells derived from definitive endoderm cells (DE). Compared with Example 1, the method adds a step of purifying definitive endoderm cells by using flow cytometry sorting technology, thereby obtaining definitive endoderm cells with higher purity (percentage of SOX17 positive cells>95%).

[0118] The specific implementation is as follows: Human embryonic stem cell H9 (SOX17-GFP reporter cell line) was cultured in E8 medium, and fresh medium was replaced every day. When the cell density reached 70-80%, it was passaged. First, it was washed twice with DPBS-EDTA, and then incubated at room temperature for 5 minutes. The DPBS-EDTA was aspirated for the third time, and E8 medium containing 5 μM of ROCK inhibitor was added. After resuspending the cells, they were passaged at a density of 1:6 and added to the well plate pre-coated with Matrigel. After the cells adhered to the wall for 1 day, they were changed to E8 medium and continued to be cultured. When the cells grew to 30-60%, differentiation began. On the first day, the first differentiation medium containing CHIR99021-HCl 5 μM and Activin A 100ng / ml was added. From the second to the third day of differentiation, CHIR99021 was removed, but Activin A was maintained to induce the formation of definitive endoderm cells. On the third day, flow cytometry was used to purify definitive endoderm cells. The purified definitive endoderm cells are cultured in MSC generation medium (day 3-5); from day 5 to day 15 of differentiation, fresh MSC generation medium is replaced every two days to induce the generation of a cell population containing mesenchymal stem cells; after day 15, the cells are passaged using a trypsin-based digestion solution. Generally, after 2-3 passages, definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) that meet the mesenchymal stem cell standards (ISCT standards, 2006) can be obtained.

[0119] The mesenchymal stem cells derived from the definitive endoderm cells prepared in Example 2 were tested, and the testing method was as follows:

[0120] Flow cytometry sorting was performed using a flow cytometer (commercially available from BD, model BD ARIR II) for purification, and cells were harvested by TrypLE (37°C, 5 minutes) and neutralized with 5% BSA. After washing with DPBS with 1% BSA, the cells were resuspended in DPBS, purified by BD ARIR II, and then the purified cells were placed in culture medium for further differentiation.

[0121] Flow cytometry was measured using a flow cytometer (commercially available from BD, model BD C6), and cells were harvested by TrypLE (37°C, 5 minutes) and neutralized with 5% FBS. After washing with DPBS with 1% BSA, the antibodies were incubated, and after washing, the cells were resuspended in DPBS and analyzed by BD C6.

[0122] Please refer to the differentiation scheme provided in this example Figure 8 For test results, please refer to Figure 9-11 .

[0123] from Figure 8It can be seen that in this example, the purified SOX17-positive definitive endoderm cells were further cultured in the subsequent stages of differentiation. Fig. 9 and Fig.10 It can be seen that the cell morphology on the 15th day is more complex, indicating that there are more types of cells at this time; after 2-3 passages, on the 24th day, the cell morphology is uniform, which is a typical spindle cell morphology of mesenchymal stem cells, indicating the formation of mesenchymal stem cells. Fig.11 It can be seen that the purified definitive endoderm-derived mesenchymal stem cells can highly express the characteristic positive biomarkers of mesenchymal stem cells (CD44, CD73, CD105) (three repeated experiments, *p<0.05).

[0124] comprehensive Figure 9-11 It can be seen that the purified (SOX17 positive cell percentage> 95%) definitive endoderm cells can be obtained by the method of this embodiment. After continuing through the third, fourth, and fifth stages, mesenchymal stem cells (DE-MSCs) generated by pure definitive endoderm cells can be obtained by this method, and these mesenchymal stem cells meet the requirements of the International Stem Cell Association (ISCT Standard, 2006) for mesenchymal stem cell standards.

[0125] Example 3

[0126] This embodiment provides a method for differentiating mesenchymal stem cells derived from definitive endoderm cells (DE). Compared with Example 2, the method further comprises the step of continuing to regulate cell signaling pathways after obtaining definitive endoderm cells, which is helpful for generating mesenchymal stem cells.

[0127] The specific implementation is as follows: Human embryonic stem cell H9 (SOX17-GFP reporter cell line) was cultured in E8 medium, and fresh medium was replaced every day. When the cell density reached 70-80%, it was passaged. First, it was washed twice with DPBS-EDTA, and then incubated at room temperature for 5 minutes. The DPBS-EDTA was aspirated for the third time, and E8 medium containing 5 μM of ROCK inhibitor was added. After resuspending the cells, they were passaged at a density of 1:6 and added to the well plate pre-coated with Matrigel. After the cells adhered to the wall for 1 day, they were changed to E8 medium and continued to be cultured. When the cells grew to 30-60%, differentiation began. On the first day, the first differentiation medium containing CHIR99021-HCl 5 μM and Activin A 100ng / ml was added. From the second to the third day of differentiation, CHIR99021 was removed, but Activin A was maintained to induce the formation of definitive endoderm cells. On the third day, flow cytometry was used to purify definitive endoderm cells. The definitive endoderm cells were cultured in MSC generation medium (day 3-5), and WNT pathway activators including CHIR99021 (5 μM) and WNT pathway activators including CHIR99021 (5 μM) / TGF pathway inhibitor SB431542 (10 μM) were added to obtain midgut / hindgut fate; from day 5 to day 15 of differentiation, fresh MSC generation medium was replaced every two days to induce the generation of a cell population containing mesenchymal stem cells; after day 15, trypsin-based digestion solution was used for passage. Generally, after 2-3 passages, definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) that meet the mesenchymal stem cell standards (ISCT standards, 2006) can be obtained.

[0128] The mesenchymal stem cells derived from the definitive endoderm cells prepared in Example 3 were tested, and the testing method was as follows:

[0129] qPCR was measured using a fluorescent quantitative PCR detector (model: Thermo Fisher QuantStudio7), total mRNA was extracted using RNAiso-plus (TAKARA, catalog number 108-95-2), and reverse transcription from mRNA to cDNA was performed using a high-capacity cDNA reverse transcription kit (AppliedBiosystems, catalog number 4368813). Real-time quantitative PCR was performed using SYBR Premix Ex Taq (TAKARA, catalog number RR420) and the Quantstudio-7 system (Applied Biosystems). The relative amount of amplified nucleotide fragments was calculated by the 2^(-ΔCt) method. The expression level was normalized to the housekeeping gene TBP and compared with undifferentiated embryonic stem cells.

[0130] Flow cytometry was measured using a flow cytometer (model: BD C6), and cells were harvested by TrypLE (37°C, 5 min) and neutralized with 5% FBS. After washing with DPBS with 1% BSA, the antibodies were incubated, and after washing, the cells were resuspended in DPBS and analyzed by BD C6.

[0131] Please refer to the differentiation scheme provided in this example Fig.12 For test results, please refer to Figure 13-Figure 15 .

[0132] from Fig.12 It can be seen that this embodiment provides a scheme for activating the Wnt pathway and inhibiting the TGF pathway on days 3-5. Fig.13 Representative qPCR analysis to test the effects of CHIR99021, SB431542, XAV939 and their combination on day 15 CD44 , CD73 , CD105 The effect of mRNA expression level. Fig.14 It can be seen that after 2-3 passages, on the 24th day, the cell morphology became uniform, a spindle-shaped cell morphology, indicating the formation of mesenchymal stem cells.

[0133] from Fig.15 It can be seen that DE-MSCs (FBS) means that DE was treated only in the presence of FBS, DE-MSCs (CHIR) means that DE was treated with CHIR99021 for an additional 2 days in the presence of FBS, and DE-MSCs (CHIR / SB) means that DE was treated with CHIR99021 and SB431542 for an additional 2 days in the presence of FBS. On day 24, all of them were positive for mesenchymal stem cell positive markers CD44, CD73, CD105, and PDGFRβ, and negative for mesenchymal stem cell negative marker CD45.

[0134] Combination Figure 13-Figure 15 It can be seen that after obtaining definitive endoderm cells, continuing to activate the Wnt pathway and inhibiting the TGF pathway is conducive to the generation of mesenchymal stem cells derived from definitive endoderm cells, and these mesenchymal stem cells meet the requirements of the International Stem Cell Society (ISCT standards, 2006) for mesenchymal stem cell standards.

[0135] Example 4

[0136] This embodiment provides a method for differentiating mesenchymal stem cells derived from definitive endoderm cells (DE). Mesenchymal stem cells can also be obtained through definitive endoderm cells via human embryonic stem cell line H1 and human induced pluripotent stem cell line NL-1.

[0137] The specific implementation is as follows: Human embryonic stem cell line H1 and human induced pluripotent stem cell line NL-1 were cultured in E8 medium, and fresh medium was replaced every day. When the cell density reached 70-80%, the cells were passaged. First, they were washed twice with DPBS-EDTA (commercially available from Cell system, catalog number 4Z0-610), then incubated at room temperature for 5 minutes, aspirated DPBS-EDTA for the third time, and added to the well plate containing 5 μM Matrigel of ROCK inhibitor. After the cells adhered to the wall for 1 day, they were replaced with E8 medium and continued to be cultured. When the cells grew to 30-60%, differentiation began. On the first day, the first differentiation medium containing CHIR99021-HCl 5 μM and Activin A 100 ng / ml was added. On the second to third day of differentiation, CHIR99021 was removed, but Activin A was maintained to induce the formation of definitive endoderm cells. On the third day, flow cytometry was used to purify definitive endoderm cells. Culture the definitive endoderm cells in MSC generation medium (day 3-5); replace with fresh MSC generation medium every two days from day 5 to day 15 of differentiation to induce the generation of a cell population containing mesenchymal stem cells; after day 15, passage the cells using a trypsin-based digestion solution. Generally, after 2-3 passages, definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) that meet the mesenchymal stem cell standards (ISCT standards, 2006) can be obtained.

[0138] Flow cytometry was measured using a flow cytometer (commercially available from BD, model BD C6), and cells were harvested by TrypLE (37°C, 5 min) and neutralized with 5% FBS. After washing with DPBS with 1% BSA, the antibodies were incubated, washed, and the cells were resuspended in DPBS and analyzed by BD C6.

[0139] Fig.16 The cell morphology of mesenchymal stem cells derived from definitive endoderm cells after NL-1 and H1 on day 24 is spindle-shaped, showing the characteristics of mesenchymal stem cells.

[0140] Fig.17 The mesenchymal stem cells derived from definitive endoderm cells after NL-1 and H1 were positive for mesenchymal stem cell biomarkers (CD44 and CD105) on day 24.

[0141] Fig.16 and Fig.17 The results showed that mesenchymal stem cells can also be obtained through the definitive endoderm via the human embryonic stem cell line H1 and the human induced pluripotent stem cell line NL-1.

[0142] Example 5

[0143] Definitive endoderm-derived mesenchymal stem cells have the potential to treat ulcerative colitis.

[0144] The differentiation method of definitive endoderm-derived mesenchymal stem cells was the same as above. After obtaining DE-MSCs (under FBS, FBS CHIR, and FBS CHIR / SB conditions), animal experiments were performed to detect the potential of DE-MSCs in the treatment of ulcerative colitis. The specific method is as follows: 2% dextran sulfate sodium (DSS) (commercially available from MP Biomedical, molecular weight 36,000-50,000) was given to mice in drinking water for 6 days to establish a colitis model. On the 3rd and 4th days after the start of DSS, 5×10 6 DE-MSCs (under FBS, FBS CHIR, and FBS CHIR / SB conditions) or human UC-MSCs or an equal volume of PBS. The body weight of the mice was measured daily from day 0 to day 14. On day 14, the mice were euthanized by CO2 asphyxiation. After necropsy, the colon was dissected from each mouse and its length was measured.

[0145] from Fig.18 It can be seen that compared with mice not injected with mesenchymal stem cells, mice injected with mesenchymal stem cells regained weight faster, indicating that the mice recovered from colitis faster. Fig.19 and Fig. 20 It can be seen that the average colon length of mice in the mesenchymal stem cell injection group was significantly longer than that of mice not injected with mesenchymal stem cells.

[0146] In summary, the differentiation method of mesenchymal stem cells derived from definitive endoderm cells provided in the present application uses a method with clear components to induce human pluripotent stem cells to differentiate into definitive endoderm cells (DE) and definitive endoderm cell (DE)-specific mesenchymal stem cells (DE-MSCs). More importantly, the combined use of WNT pathway activators, WNT pathway inhibitors and TGF pathway inhibitors to treat definitive endoderm cells (DE) for a short time will help these cells acquire the fate of midgut cells, hindgut cells and foregut cells; the above mesenchymal stem cells derived from definitive endoderm cells via human pluripotent stem cells are collectively referred to as DE-MSCs. DE-MSCs obtained through the midgut / hindgut cell fate pathway are similar to MSCs derived from the small and large intestines of the human body, and are suitable for the treatment of digestive tract-related diseases represented by colitis and Crohn's disease. The present application provides an in vitro differentiation method for efficiently preparing mesenchymal stem cells derived from definitive endoderm derived from human pluripotent stem cells. Moreover, these DE-MSCs can secrete anti-inflammatory factors under in vitro stimulation conditions. Furthermore, these DE-MSCs injected intraperitoneally have the potential to treat colitis in mice.

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for differentiating mesenchymal stem cells derived from definitive endoderm cells, characterized in that: It includes: The first differentiation stage: culturing in a first differentiation medium containing a WNT pathway activator and Activin A for 1-2 days to induce human pluripotent stem cells to differentiate into mesendoderm cells, wherein the human pluripotent stem cells include at least one of human embryonic stem cells and human induced pluripotent stem cells, and the human embryonic stem cells are stem cells separated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development; The second differentiation stage: culturing in a second differentiation medium containing Activin A but without WNT pathway activators for 2-8 days to induce the formation of definitive endoderm cells; The third differentiation stage: culturing in MSC generation medium for 2-4 days to induce the definitive endoderm cells to acquire the fate of midgut cells, hindgut cells and foregut cells; Fourth differentiation stage: continue to culture in MSC generation medium for 8-14 days to induce the generation of cell populations containing mesenchymal stem cells; Fifth differentiation stage: Digestion and passage are performed 2-3 times to obtain mesenchymal stem cells derived from definitive endoderm cells.

2. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 1, characterized in that: After the second differentiation stage is completed, the method further comprises purifying the definitive endoderm cells by using flow cytometry sorting technology.

3. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 2, characterized in that: Purifying the definitive endoderm cells comprises: harvesting the cells by using a cell digestion solution, neutralizing the cells with a neutralizing solution, washing the cells, resuspending the cells, and purifying the cells by using a flow cytometer.

4. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 1 or 2, characterized in that: A cell signaling pathway regulator is added to the MSC generation medium in the third differentiation stage.

5. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 4, characterized in that: The cell signaling pathway regulator includes at least one of a WNT pathway activator, a WNT pathway inhibitor, and a TGF pathway inhibitor.

6. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 5, characterized in that: The WNT pathway activator includes at least one of CHIR99021, CHIR99021-HCl, Wnt3a ligand, Wnt5a ligand and BIO; the WNT pathway inhibitor includes at least one of XAV939, IWP2 and IWR-1; the TGF pathway inhibitor includes one of SB431542, RepSox, SD-208, GW788388, A-77-01 and A-83-01.

7. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 6, characterized in that: The WNT pathway activator is CHIR99021 at a treatment concentration of 1-5 μM.

8. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 6, characterized in that: The WNT pathway inhibitor is XAV939 at a treatment concentration of 1-10 μM.

9. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 6, characterized in that: The TGF pathway inhibitor is SB431542 at a treatment concentration of 1-10 μM.

10. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 1, characterized in that: The first differentiation medium is obtained by adding the WNT pathway activator and the Activin A to the basic components, and the second differentiation medium is obtained by adding the Activin A to the basic components.

11. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 10, characterized in that: The WNT pathway activator is CHIR99021-HCl at a treatment concentration of 1-5 μM, and the added amount of Activin A is 10-100 ng / ml.

12. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 10, characterized in that: The basic components include DMEM / F12, L-ascorbic acid-2-magnesium phosphate 60-70 mg / L, sodium selenite 10-15 μg / L, transferrin 8-12 μg / ml, Chemical defined lipid concentrate 1× and penicillin-streptomycin 1%.

13. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 1, characterized in that: The components of the MSC generation medium when it does not contain serum include αMEM, L-ascorbic acid-2-phosphate magnesium 60-70 mg / L, sodium selenite 10-15 μg / L, insulin 8-12 μg / ml, FGF2 90-110 ng / ml, B-27™ Supplement 1×, GlutaMAX™ Supplement 1×, NEAA 1× and penicillin-streptomycin 1%; or, The components of the MSC generation medium when containing serum include αMEM, FBS 10%-20%, L-ascorbic acid-2-phosphate magnesium 60-70 mg / L, sodium selenite 10-15 μg / L, insulin 8-12 μg / ml, FGF2 90-110 ng / ml, B-27™Supplement 1×, GlutaMAX™ Supplement 1×, NEAA 1× and penicillin-streptomycin 1%.

14. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 1, characterized in that: The human embryonic stem cells include human embryonic stem cells H1 or human embryonic stem cells H9.

15. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 1, characterized in that: The human induced pluripotent stem cells include human induced pluripotent stem cells NL-1.

16. The method for differentiating mesenchymal stem cells derived from definitive endoderm cells according to claim 1, characterized in that: The human pluripotent stem cells are differentiated at a cell density of 30-60%.

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