CN115232788A - Mesenchymal stem cell derived from definitive endoderm cell and differentiation method and application thereof - Google Patents
Mesenchymal stem cell derived from definitive endoderm cell and differentiation method and application thereof Download PDFInfo
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- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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Abstract
The invention discloses mesenchymal stem cells derived from definitive endoderm cells, and a differentiation method and application thereof, and relates to the technical field of mesenchymal stem cells. The differentiation method comprises the following steps: inducing differentiation of human pluripotent stem cells into mesendoderm cells by culturing for 1-2 days in a first differentiation medium containing a WNT pathway activator and Activin A; inducing the formation of definitive endoderm cells (DE) by culturing for 2-8 days in a second differentiation medium without WNT pathway activator and containing Activin A; culturing in MSC generating culture medium for 2-4 days to obtain cell fate of midgut, hindgut and foregut; culturing in MSC generating culture medium for 8-10 days to induce and generate cell population containing mesenchymal stem cells; digesting and passaging for 2-3 times to obtain mesenchymal stem cells (DE-MSCs) from the definitive endoderm cells. It is capable of secreting anti-inflammatory factors under in vitro stimulation conditions and has the potential to treat colitis.
Description
Technical Field
The invention relates to the technical field of mesenchymal stem cells, in particular to mesenchymal stem cells derived from definitive endoderm cells, and a differentiation method and application thereof.
Background
At present, the traditional therapies using chemical drugs, biological drugs and surgical therapies as the support still can not solve many disease problems, and regenerative medicine using stem cell technology as the core is gradually applied to clinical application and shows great potential. Mesenchymal Stem Cells (MSCs) are characterized by low immunogenicity (i.e. low expression of major histocompatibility complex MHC I in MSCs and no expression of MHC II in MSCs) compared to other types of stem cells, and therefore MSCs are more suitable for allogeneic use and have become ideal candidates for cell therapy.
Mesenchymal Stem Cells (MSCs) have a variety of therapeutic potential for tissue engineering and regenerative medicine, can be directly differentiated into tissue-specific cells to repair wounds and can stimulate tissue repair by releasing paracrine forms of anti-inflammatory cytokines, anti-apoptotic factors, and growth factors, and can be used to treat a variety of diseases, including inflammatory diseases and autoimmune diseases.
In addition, it is described in the literature that MSCs can treat diseases such as respiratory diseases, digestive diseases, liver diseases, pancreatitis, and colorectal diseases.
Despite the positive results obtained by MSCs in the treatment of respiratory and digestive disorders, and the safety and efficacy of MSCs therapy as demonstrated by a number of clinical trials, the results and conclusions of these studies are still very different. The most reasonable interpretation is: 1) The source of MSCs donors is too extensive, traditionally, mesenchymal stem cells are derived from human tissues, the main sources include bone marrow and fat, MSCs can also be obtained by separating tissues such as umbilical cord blood, placenta, amnion, dental pulp and the like, from the embryonic development perspective, bone marrow, fat, dental pulp, umbilical cord, placenta, amnion and the like are derived from mesoderm, ectoderm or trophoblast, while MSCs derived from mesoderm, ectoderm or trophoblast are not necessarily suitable for endoderm (progenitor cells of respiratory tract organs and digestive tract organs) related diseases; 2) There are also large differences in the culture protocols for MSCs from different donor sources. It is therefore essential to generate homogeneous and germ layer matched MSCs for therapeutic use.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide mesenchymal stem cells derived from definitive endoderm cells, and a differentiation method and application thereof.
The invention is realized by the following steps:
in a first aspect, the present invention provides a method of differentiating definitive endoderm cell-derived mesenchymal stem cells, comprising:
a first differentiation stage: culturing in a first differentiation medium containing a WNT pathway activator and Activin A for 1-2 days to induce differentiation of human pluripotent stem cells into mesendoderm cells;
and a second differentiation stage: inducing the formation of definitive endoderm cells (DE) by culturing for 2-8 days in a second differentiation medium without WNT pathway activator and containing Activin A;
a third differentiation stage: culturing in MSC production culture medium for 2-4 days to induce the definitive endoderm cell to obtain fates of midgut cell, hindgut cell and foregut cell;
and a fourth differentiation stage: continuously culturing in the MSC generating culture medium for 8-14 days to induce and generate a cell population containing the mesenchymal stem cells;
a fifth separation stage: digesting and passaging for 2-3 times to obtain mesenchymal stem cells (DE-MSCs) derived from definitive endoderm cells.
In a second aspect, the present invention provides a definitive endoderm cell-derived mesenchymal stem cell differentiated by the method of producing a definitive endoderm cell-derived mesenchymal stem cell from a human pluripotent stem cell according to any one of the preceding embodiments.
In a third aspect, the present invention provides the use of definitive endoderm cell-derived mesenchymal stem cells according to the previous embodiments in the manufacture of a medicament for the treatment of a gut-related disease.
In a fourth aspect, the present invention provides a mesenchymal stem cell differentiated cell, which is obtained by in vitro differentiation of a mesenchymal stem cell derived from a definitive endoderm cell according to the above-described embodiment;
preferably, the differentiated cells of the mesenchymal stem cells include at least one of adipocytes, chondrocytes, and osteoblasts.
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, comprising reagents for implementing the method for differentiating definitive endoderm cell-derived mesenchymal stem cells according to the foregoing embodiments.
The invention has the following beneficial effects:
the differentiation method of mesenchymal stem cells from definitive endoderm cells provided by the application adopts a culture medium with clear components to induce hPSC to differentiate into definitive endoderm cells (DE) and mesenchymal stem cells from definitive endoderm cells (DE-MSCs). More importantly, the cells can be allowed to acquire midgut, hindgut and foregut cell fates by different induction modalities. DE-MSCs obtained by the way of foregut cell fate are similar to human lung, liver and pancreas derived MSCs, and are suitable for treating body lung, liver and pancreas related diseases represented by COVID-19, liver cirrhosis, pancreatitis and the like; DE-MSCs obtained by the way of cell fate of midgut/hindgut are similar to MSCs derived from human small intestine and large intestine, and are suitable for treating intestinal tract-related diseases represented by colitis and Crohn's disease. The application provides an in vitro differentiation method for efficiently preparing definitive endoderm-derived mesenchymal stem cells derived from human pluripotent stem cells. And the DE-MSCs can secrete anti-inflammatory factors under the condition of in vitro stimulation, and furthermore, the DE-MSCs injected by the abdominal cavity have the capacity of treating colitis mice.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a scheme of differentiation of definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) as provided in example 1;
FIG. 2 is the change in cell morphology (upper panel) over time and the change in expression of the definitive endoderm cell marker SOX17 (lower panel) over time from day 0 to day 24 in example 1;
FIG. 3 is the change of the expression levels of CD44, CD73, CD105mRNA, genes characteristic of SOX17 mRNA and mesenchymal stem cells, with time course in example 1;
figure 4 is the percentage of mesenchymal stem cell markers CD44, CD73, CD105, CD45 positive cells at day 24 in example 1;
FIG. 5 is the percentage of definitive endoderm cell markers SOX17 positive cells and the percentage of mesenchymal stem cell markers CD44, CD73 and CD105 positive cells in example 1;
FIG. 6 is a graph of the total number of cells of definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) as a function of time course in example 1;
FIG. 7 shows the further differentiation of definitive endoderm derived mesenchymal stem cells (DE-MSCs) into adipocytes (stained red with oil red O dye), chondrocytes (stained blue with Alcian blue dye), osteocytes (stained black with Von Kossa dye) in example 1;
FIG. 8 is a schematic representation of the differentiation protocol for definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) provided in example 2;
FIG. 9 shows SOX 17-positive DE cells purified in example 2 (only the first 48% of DE cells expressing the highest intensity were selected and the purified cells were returned to MSC-producing conditions to induce further formation of DE-MSCs);
FIG. 10 shows cell morphology at day 15 and day 24 in example 2;
FIG. 11 is the percentage of CD44, CD73, CD105 positive cells at day 24 in example 2;
FIG. 12 is a schematic representation of the differentiation protocol for definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) provided in example 3;
FIG. 13 is an RT-qPCR analysis to test the effect of CHIR99021, SB431542, XAV939 and combinations thereof on day 15 CD44, CD73, CD105mRNA expression levels in example 3;
FIG. 14 is the cell morphology of DE-MSCs (FBS), DE-MSCs (CHIR/SB) on day 24 in example 3;
FIG. 15 is the percentage of CD44, CD73, CD105, PDGFR β, CD45 positive cells on day 24 for DE-MSCs (FBS), DE-MSCs (CHIR/SB) in example 3;
FIG. 16 is the cell morphology of definitive endoderm-derived mesenchymal stem cells (DE-MSCs) on day 24 of human induced pluripotent stem cells NL-1 and human embryonic stem cells H1 in example 4;
FIG. 17 shows the percentage of CD44, CD105 positive cells at day 24 of definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) derived from human induced pluripotent stem cells NL-1 and human embryonic stem cells H1 in example 4;
FIG. 18 is the body weight changes from day 0 to day 14 in the mice of the healthy group, the mice of the DSS + PBS + DE-MSCs (FBS) group, the mice of the DSS + PBS + DE-MSCs (CHIR/SB) group, and the mice of the DSS + PBS + UC-MSCs group in example 5;
FIG. 19 is the colon length at day 14 of the mice of the healthy group, the mice of the DSS + PBS + DE-MSCs (FBS) group, the mice of the DSS + PBS + DE-MSCs (CHIR/SB) group, and the mice of the DSS + PBS + UC-MSCs group in example 5;
FIG. 20 is an image of the colon at day 14 of the healthy group mouse, the DSS + PBS + DE-MSCs (FBS) group mouse, the DSS + PBS + DE-MSCs (CHIR/SB) group mouse, and the DSS + PBS + UC-MSCs group mouse in example 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a differentiation method of mesenchymal stem cells derived from definitive endoderm cells, which comprises the following steps: human pluripotent stem cells are induced to differentiate into definitive endoderm cells (DE), definitive endoderm cells are induced to differentiate into Mesenchymal Stem Cells (MSCs), and mesenchymal stem cells derived from definitive endoderm cells derived from human pluripotent stem cells are collectively referred to as DE-MSCs.
Specifically, the differentiation method provided by the present application comprises the following steps:
s1, culturing and passaging the human pluripotent stem cells, and beginning to differentiate when the cells grow to 30-60%.
Human pluripotent stem cells were cultured in E8 medium, and fresh E8 medium was changed every day, and passaging was performed when the cell density reached 70-80%. During passage, firstly, washing the human pluripotent stem cells with DPBS-EDTA for 2-3 times, then incubating for 4-10 minutes at room temperature, sucking the DPBS-EDTA, adding a dry maintenance medium containing 5 mu M ROCK inhibitor, after resuspending the cells, performing passage at a density of 1.
Wherein, the components of the E8 culture medium comprise: DMEM/F12 (basal medium, commercially available from ThermoFisher under the trade designation 11330-032), L-ascorbic acid-2-magnesium phosphate 60-70mg/L, sodium selenite 10-15. Mu.g/L, transferrin 8-12. Mu.g/ml, insulin 8-12. Mu.g/ml, FGF 2-110 ng/ml, TGF beta 1-2ng/ml.
The human pluripotent stem cells comprise at least one of human embryonic stem cells and human induced pluripotent stem cells; preferably, the human embryonic stem cells include human embryonic stem cells H1 or human embryonic stem cells H9; the human induced pluripotent stem cell includes a human induced pluripotent stem cell NL-1.
Human embryonic stem cells H1 and H9 herein were purchased from WiCell Research Institute, inc. (Madison, wis., USA, http:// www.Wicell. Org.) and human induced pluripotent stem cell NL-1 was the cell line deposited in this laboratory (hPScreg accession number CRMi 003-A).
S2, differentiation stage.
(1) A first differentiation stage.
Human pluripotent stem cells were induced to differentiate into mesendoderm by culturing for 1-2 days in a first differentiation medium containing WNT pathway activator and Activin a.
WNT pathway activator comprises at least one of CHIR99021, CHIR99021-HCl, wnt3a ligand, wnt5a ligand and BIO; the treatment concentration of CHIR99021 or CHIR99021-HCl is 1-5. Mu.M.
Activin A is a secreted protein of Transforming Growth Factor (TGF) beta family, can control embryonic axis to develop 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.
In the present application, a WNT pathway activator and Activin a are used in combination to exert a more significant effect of inducing differentiation into mesendoderm cells.
The term "first differentiation medium" used herein refers to a medium that contains WNT pathway activator and Activin a and is used as a basis for inducing differentiation of stem cells. That is, in the present application, the first differentiation medium is obtained by adding WNT pathway activator and said Activin a as basic components. Wherein the addition amount of WNT pathway activator is 1-5 μ M, and the addition amount of Activin A is 10-100ng/ml.
Specifically, the basic components include DMEM/F12, L-ascorbic acid-2-magnesium phosphate 60-70mg/L, sodium selenite 10-15. Mu.g/L, transferrin 8-12. Mu.g/ml, chemical defined lipid concentrate 1X and streptomycin 1%.
Culturing for 1 to 2 days, i.e., the culture time is calculated from the 0 th day of differentiation, the "0 th day" herein refers to the initiation point of inducing differentiation, i.e., 0 to 24 hours, and culturing for 1 to 2 days is understood as culturing with the first differentiation medium containing WNT pathway activator and Activin a at any time within 0 to 48 hours after the initiation of differentiation, preferably, the differentiation culture time is 1 day.
Specifically, the differentiation medium used in the present application may contain WNT pathway activator and Activin a at any time from day 1 to day 2, specifically at any time point or any time period from day 0, day 0.5, day 1, day 1.5, day 2. It should be noted that, when the time period of the differentiation medium containing the specific reagent is a time period, the culture medium may be selectively replaced for several times in the time period so that the differentiation medium contains the specific reagent, for example, the time period of 1 to 2 days may be replaced with a new culture medium every day, every 10 hours, or every 20 hours, so that the process of inducing differentiation is more stable and effective, and similar cases are understood in the same way and are not described again.
(2) And (5) a second differentiation stage.
Definitive endoderm cells were induced by culturing for 2-8 days in a second differentiation medium without WNT pathway activator and with Activin a.
The present application removes WNT pathway activators in the second differentiation stage, and only adds Activin a as the second differentiation medium to the base component for culture, at which time, definitive endoderm cells (DE) can be further induced to form. In general, high-purity definitive endoderm cells (DE) can be obtained by treating Activin A (10-100 ng/ml) for two days, but high-purity definitive endoderm cells (DE) can also be obtained by prolonging the treatment time of Activin A (10-100 ng/ml) to 4 days, 6 days and 8 days, and then DE-MSCs can still be obtained according to subsequent steps. Thus, the second differentiation stage in the present application may be cultured for 2-8 days, preferably 2 days.
Specifically, the base components included DMEM/F12 (which is a basal medium, commercially available from ThermoFisher, cat # 11330-032), L-ascorbic acid-2-magnesium phosphate (working concentration 60-70 mg/L), sodium selenite (working concentration 10-15. Mu.g/L), transferrin (working concentration 8-12. Mu.g/ml), chemical-defined lipid concentrate (chemically-defined lipid concentrate, commercially available from ThermoFisher, cat # 11905031) 1 Xand streptomycin (working concentration 1%).
Preferably, after differentiation to obtain definitive endoderm cells (DE), flow cytometric sorting is used to purify definitive endoderm cells (DE). Purification of DE includes: the cells were harvested by cell digest and neutralized with cell neutralization solution, washed, resuspended, and purified by flow cytometry.
(3) And a third differentiation stage.
Culturing in MSC production medium for 2-4 days to induce the definitive endoderm cells to obtain the fate of midgut cells, hindgut cells and foregut cells. Wherein the midgut cells are characterized by expressing the biomarker PDX1, the hindgut cells are characterized by expressing the biomarker CDX2, and the foregut cells are characterized by expressing the biomarker SOX 2.
A cellular signaling pathway modulator, which herein includes at least one of a WNT pathway activator, a WNT pathway inhibitor, and a TGF pathway inhibitor, may be added to the MSC production medium at the third differentiation stage.
Wherein the WNT pathway activator comprises at least one of CHIR99021, CHIR99021-HCl, wnt3a ligand, wnt5a ligand and BIO; the treatment concentration of WNT pathway activator is 1-5 muM; WNT pathway inhibitor comprises at least one of XAV939, IWP2 and IWR-1, and the treating concentration of WNT pathway inhibitor is 1-10 μ M; the TGF pathway inhibitor comprises one of SB431542, repsox, SD-208, GW788388, A-77-01 and A-83-01; treatment concentrations of TGF pathway inhibitor were 1-10. Mu.M.
The components of MSC production culture medium without serum comprise alpha MEM, L-ascorbic acid-2-magnesium phosphate 60-70mg/L, sodium selenite 10-15 μ g/L, insulin 8-12 μ g/ml, FGF 2-110 ng/ml, B-27TMsupplement 1X, glutaMAXtMSsupplement 1X, NEAA 1X and streptomycin 1%; or when the MSC production culture medium contains serum, the components comprise alpha MEM, 10-20% of FBS, 60-70mg/L of L-ascorbic acid-2-magnesium phosphate, 10-15 mu g/L of sodium selenite, 8-12 mu g/ml of insulin, 2-110 ng/ml of FGF, B-27TMsupplement 1X, glutaMAXTM Supplement 1X, NEAA 1X and 1% of streptomycin.
(4) The fourth decomposition stage
Continuously culturing in MSC generating culture medium for 8-10 days to induce and generate cell population containing mesenchymal stem cells; in the fourth differentiation stage, cells are cultured only by using the MSC generation culture medium without adding a cell signaling pathway regulator, and the liquid is changed every two days to induce and generate a cell population containing the mesenchymal stem cells.
(5) Fifth fractionation stage
Performing conventional digestion passage with trypsin digestion solution, and after passage for 2-3 times, obtaining mesenchymal stem cells (DE-MSCs) derived from definitive endoderm cells.
The gene expression profiles of mesenchymal stem cells (DE-MSCs) derived from definitive endoderm cells obtained by the differentiation method are similar to the endoderm organ gene expression profile; furthermore, the DE-MSCs injected by the abdominal cavity have the potential of treating mouse colitis, and can be widely applied to the preparation of medicaments for treating intestinal tract related diseases, particularly, the intestinal tract related diseases comprise but are not limited to colitis, respiratory diseases and digestive tract inflammatory diseases.
Correspondingly, the application also provides a kit for inducing the human pluripotent stem cells to generate mesenchymal stem cells derived from the definitive endoderm cells, which comprises a reagent for realizing the differentiation method of the mesenchymal stem cells derived from the definitive endoderm cells.
In addition, the application also provides a mesenchymal stem cell differentiated cell which is formed by in vitro differentiation of the mesenchymal stem cell derived from the definitive endoderm; the mesenchymal stem cell differentiated cell includes at least one of an adipocyte, a chondrocyte and an osteoblast.
The materials and reagents used in the following examples were derived from:
CHIR99021 (CT 99021) HCl, all known as WNT pathway activators, commercially available from seleckchem under cat number S2924;
activin A, all known as a transforming growth factor superfamily member, commercially available from R & D Systems under the accession number 338-AC-500/CF;
TGF β, collectively known as transforming growth factor β, commercially available from R & D Systems under Cat No. 240-B-500;
FGF2, all known as fibroblast growth factor 2, commercially available from Peprotech, cat # 100-18C:
XAV939, all known as WNT pathway inhibitors, commercially available from Selleckchem under cat number S1180;
SB432542, all known as TGF pathway inhibitors, commercially available from Selleckchem under cat # S1067;
DMEM/F12 is a basal medium, commercially available from ThermoFisher under the catalog number 11330-032;
chemical defined lipid concentrate is a chemically defined lipid concentrate, commercially available from ThermoFisher under cat No. 11905031;
alpha MEM was a basal medium, commercially available from ThermoFisher under Cat number 12571063;
FBS, fully known as fetal bovine serum, commercially available from Gibco under cat # 10437028;
B-27 TM supplement, full name B-27 TM Supplements, commercially available from ThermoFisher, cat No. 17504044, at an initial concentration of 50 ×;
GlutaMAX TM supplement, known as GlutaMAX TM Supplement, commercially available from Gibco, cat # 35050061, at an initial concentration of 100 ×;
MEM Non-Essential Amino Acids solutions (NEAA), all known as MEM nonessential Amino Acids solutions, commercially available from Gibco under the reference number 11140050, at an initial concentration of 100 ×;
DPBS-EDTA, available from Cell system, cat # 4Z0-610;
human embryonic stem cell H1: purchased from WiCell Research Institute, inc. (Madison, wis., USA, http:// www. Cell. Org.);
human embryonic stem cells H9: purchased from WiCell Research Institute, inc. (Madison, wis., USA, http:// www.wicell. Org.);
human induced pluripotent stem cell NL-1: the cell line deposited in this laboratory was named CRMi003-A in the hPSreg human induced pluripotent stem cell registry (https:// hpscereg. Eu).
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a method for differentiating mesenchymal stem cells (DE-MSCs) derived from definitive endoderm cells (DE), specifically using a method of stepwise in vitro differentiation of stem cells.
The concrete implementation is as follows: human embryonic stem cells H9 (SOX 17-GFP reporter cell line, SOX17 being a specific biomarker for definitive endoderm cells, SOX17-GFP reporter cell line exhibiting green fluorescence upon differentiation into definitive endoderm cells, and therefore, the formation of definitive endoderm cells was observed by fluorescence microscopy or flow cytometry) were cultured in E8 medium, and the medium was replaced with fresh medium every day and passaged when the cell density reached 70-80%. First, 2 washes with DPBS-EDTA were performed, followed by incubation at room temperature for 5 minutes, aspiration of DPBS-EDTA, and addition of E8 medium containing ROCK inhibitor 5. Mu.M. After resuspension of the cells, they were passaged at a density of 1. After the cells are attached to the wall for 1 day, the E8 culture medium is changed for continuous culture. When the cells grow to 30-60%, differentiation is started, and the first day, the cells are cultured by using a first differentiation medium which is prepared by adding CHIR99021-HCl 5 mu M and Activin A100ng/ml to basic components. On days 2 to 3 of differentiation, definitive endoderm cells were induced by culturing in a second differentiation medium supplemented with 100ng/ml of Activin A as a base ingredient. Changing fresh MSC production medium from day 3 to day 5 of differentiation; changing fresh MSC to generate a culture medium from the 5 th day to the 15 th day of differentiation, changing liquid once every two days, and inducing to generate a cell population containing mesenchymal stem cells; after 15 days, passage using trypsin-based digestate, typically after 2-3 passages, definitive endoderm-derived mesenchymal stem cells (DE-MSCs) meeting the mesenchymal stem cell standard (ISCT standard, 2006) were obtained.
Wherein, the components of the E8 culture medium comprise: DMEM/F12 (basal medium, commercially available from ThermoFisher, cat # 11330-032), L-ascorbic acid-2-magnesium phosphate 64mg/L, sodium selenite 13.6. Mu.g/L, transferrin 10. Mu.g/ml, insulin 10. Mu.g/ml, FGF2 100ng/ml, TGF beta 1.74ng/ml.
The basic components of the first differentiation medium and the second differentiation medium comprise: DMEM/F12, L-ascorbic acid-2-magnesium phosphate 64mg/L, sodium selenite 13.6. Mu.g/L, transferrin 10. Mu.g/ml, chemical defined lipid concentration 1X and streptomycin 1%.
The components of the MSC production medium (containing serum) include: alpha MEM, 10-20% of FBS, 64mg/L of L-ascorbic acid-2-magnesium phosphate, 13.6 mu g/L of sodium selenite, 10 mu g/ml of transferrin, and pancreatic islet10 ug/ml of a peptide, 100ng/ml of FGF2, B-27 TM Supplement 1×、GlutaMAX TM Supplement 1X, MEM Non-Essential Amino Acids Solution 1X and streptomycin 1%.
The components of the MSC production medium (serum free) include: alpha MEM, L-ascorbic acid-2-magnesium phosphate 64mg/L, sodium selenite 13.6. Mu.g/L, transferrin 10. Mu.g/ml, insulin 10. Mu.g/ml, FGF2 100ng/ml, B-27 TM Supplement 1×、GlutaMAX TM Supplement 1X, MEM Non-Essential Amino Acids Solution 1X and streptomycin 1%.
The mesenchymal stem cells derived from the definitive endoderm cells prepared in example 1 are detected by the following method:
qPCR was measured using a fluorescent quantitative PCR detector (commercially available from ThermoFisher, model QuantStudio 7), total mRNA was extracted using RNAioso-plus (commercially available from TAKARA, cat # 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, cat # 4368813). Real-time quantitative PCR was performed using SYBR Premix Ex Taq (commercially available from TAKARA, cat # RR 420) and Quantstudio-7 system. The relative amount of amplified nucleotide fragments was calculated by the 2^ (- Δ Ct) method. Expression levels were normalized to housekeeping gene TBP and compared to undifferentiated human embryonic stem cells.
Immunofluorescence was measured using an immunofluorescence analyzer (commercially available from ThermoFisher, model EVOS Cell Imaging System FL) and cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, washed with 1 XPBS (5 minutes, 3 times each), disrupted with 0.5% Triton X-100 for 20 minutes, and then washed with 1 fold amount of PBS (5 minutes, 3 times each). Nuclei were stained with Hoechst33342 (commercially available from Abnova under stock number U0334) for 20 minutes at room temperature. The cells were then rinsed with 1 × PBS (5 min, 3 times each), incubated with the antibody overnight at 4 ℃, rinsed with 1 × PBS, incubated with fluorescent secondary antibody for 2 hours, and finally rinsed with 1-fold amount of PBS before imaging.
Flow cytometry was measured using a flow cytometer (commercially available from BD, model BD C6), cells were harvested by TrypLE (37 ℃,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.
Referring to fig. 1, the differentiation protocol provided in this example is shown in fig. 2-7.
It can be seen from figure 2 that at day 3 and day 5, the definitive endoderm-specific protein SOX17 is highly expressed, indicating that high purity definitive endoderm cells have been obtained through the first and second stages of differentiation. Spindle cells were observed at day 24 and their SOX17 negatives, indicating that these definitive endoderm cells have left the original cell fate and have differentiated into cells with mesenchymal stem cell morphology (spindle).
From the results of fig. 3, it can be seen that the definitive endoderm cells have left the original cell fate and thus differentiated into cells having mesenchymal stem cell gene characteristics (CD 44, CD73, CD 105) from the results of gradually decreasing the expression level of SOX17 mRNA on days 3, 5, 10, 15, 18 (P1), 21 (P2), 24 (P3), and gradually increasing the expression level of CD44, CD73, CD105mRNA, which is a characteristic gene of mesenchymal stem cells (three repeated experiments, P < 0.05).
From figure 4 it can be seen that mesenchymal stem cells derived via definitive endoderm cells were able to highly express positive biomarkers (CD 44, CD73, CD 105) characteristic of mesenchymal stem cells while not expressing negative biomarkers (CD 45) characteristic of mesenchymal stem cells (three replicates,. P < 0.05).
As can be seen from fig. 5, the percentage of SOX17 positive cells gradually decreased on days 0, 3, 5, 10, 15, 18 (P1), 21 (P2), 24 (P3), while the percentage of CD44, CD73 and CD105 positive cells gradually increased on days 18 (P1), 21 (P2), 24 (P3) (three replicates,. P < 0.05).
FIG. 6 shows that mesenchymal stem cells generated in vitro by definitive endoderm cells have in vitro proliferation ability.
FIG. 7 shows that mesenchymal stem cells generated in vitro by definitive endoderm cells have the ability to continue differentiating into adipocytes, chondrocytes, and osteoblasts in vitro.
In combination with FIGS. 2-7, it can be seen that high purity definitive endoderm cells can be obtained by the methods of this example during the first and second stages of differentiation. After continuing through the third, fourth and fifth stages, mesenchymal stem cells generated from definitive endoderm cells can be obtained by the method of this example, and these mesenchymal stem cells meet the requirements of international association of stem cells (ISCT standard, 2006) on the mesenchymal stem cells specification.
Example 2
This example provides a differentiation method of mesenchyma stem cell derived from Definitive Endoderm (DE), which increases the steps of purifying definitive endoderm cells by flow cytometry sorting compared to example 1, and thus can obtain more highly pure definitive endoderm cells (SOX 17 positive cell percentage > 95%).
The concrete implementation is as follows: human embryonic stem cells H9 (SOX 17-GFP reporter cell line) were cultured in E8 medium, with daily changes of fresh medium and passaging when the cell density reached 70-80%. First 2 washes with DPBS-EDTA, then incubate for 5 minutes at room temperature, aspirate DPBS-EDTA 3 times, and add E8 medium containing 5. Mu.M of ROCK inhibitor. After resuspension of the cells, they were passaged at a density of 1. After the cells are attached to the wall for 1 day, the E8 culture medium is changed for continuous culture. When the cells grew to 30-60% differentiation was initiated, the first day a first differentiation medium containing CHIR99021-HCl 5. Mu.M and Activin A100ng/ml was added. Day 2 to day 3 of differentiation, CHIR99021 was removed, but Activin a was maintained to induce the formation of definitive endoderm cells. On day 3, definitive endoderm cells were purified using flow cytometric sorting techniques. Culturing the purified definitive endoderm cells in an MSC production culture medium (3-5 days); changing fresh MSC to generate a culture medium from the 5 th day to the 15 th day of differentiation, changing liquid once every two days, and inducing to generate a cell population containing mesenchymal stem cells; after 15 days, passage using trypsin-based digestate, typically after 2-3 passages, definitive endoderm-derived mesenchymal stem cells (DE-MSCs) meeting the mesenchymal stem cell standard (ISCT standard, 2006) were obtained.
The mesenchymal stem cells from the definitive endoderm cells prepared in example 2 are detected by the following method:
flow cytometry was purified using a flow cytometer (commercially available from BD, model BD ARIR II), cells were harvested by TrypLE (37 ℃,5 minutes) and neutralized with 5% bsa. After washing with DPBS with 1% bsa, cells were resuspended in DPBS, purified by BD ARIR II, and then the purified cells were put on the medium to continue differentiation.
Flow cytometry was measured using a flow cytometer (commercially available from BD, model BD C6), cells were harvested by TrypLE (37 ℃,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.
Referring to fig. 8, the differentiation protocol provided in this example, and the assay results are shown in fig. 9-11.
As can be seen from fig. 8, in this example, the purified SOX 17-positive definitive endoderm cells were further subjected to the subsequent differentiation culture on day 3, and as can be seen from fig. 9 and 10, the cell morphology on day 15 was more complex, indicating that the cell types were more in this case; after 2-3 passages, the cell morphology is classified as uniform at day 24, and is a typical fusiform cell morphology of the mesenchymal stem cells, and the formation of the mesenchymal stem cells is prompted. As can be seen from fig. 11, the mesenchymal stem cells derived from the purified definitive endoderm cells can highly express mesenchymal stem cell characteristic positive biomarkers (CD 44, CD73, CD 105) (three repeated experiments, p < 0.05).
Taken together in FIGS. 9-11, it can be seen that purified (SOX 17-positive cell percentage > 95%) definitive endoderm cells can be obtained by the method of this example. After the third, fourth and fifth stages, mesenchymal stem cells (DE-MSCs) generated by pure definitive endoderm cells can be obtained by the method, and the mesenchymal stem cells meet the requirements of the international association of stem cells (ISCT standard, 2006) on the mesenchymal stem cell standard.
Example 3
This example provides a method of differentiating definitive endoderm cell (DE) -derived mesenchymal stem cells, which compared to example 2, increased continued modulation of cell signaling pathways after definitive endoderm cells were obtained, contributing to the generation of mesenchymal stem cells.
The concrete implementation is as follows: human embryonic stem cells H9 (SOX 17-GFP reporter cell line) were cultured in E8 medium, replaced with fresh medium every day, and passaged when the cell density reached 70-80%. First, 2 washes with DPBS-EDTA were performed, followed by incubation at room temperature for 5 minutes, 3 rd aspiration of DPBS-EDTA and addition of E8 medium containing ROCK inhibitor 5. Mu.M. After resuspending the cells, they were passaged at a density of 1. And E8 culture medium is changed for further culture 1 day after the cells are attached to the wall. When the cells grew to 30-60% differentiation was initiated, the first day a first differentiation medium containing CHIR99021-HCl 5. Mu.M and Activin A100ng/ml was added. Day 2 to day 3 of differentiation, CHIR99021 was removed, but Activin a was maintained to induce the formation of definitive endoderm cells. On day 3, definitive endoderm cells were purified using flow cytometric sorting techniques. Culturing definitive endoderm cells in MSC production medium (day 3-5), adding WNT pathway activator including CHIR99021 (5 μ M), WNT pathway activator including CHIR99021 (5 μ M)/TGF pathway inhibitor SB431542 (10 μ M) to obtain midgut/hindgut fate; changing fresh MSC to generate a culture medium from the 5 th day to the 15 th day of differentiation, changing liquid once every two days, and inducing to generate a cell population containing mesenchymal stem cells; after 15 days, passage using trypsin-based digestate, typically after 2-3 passages, definitive endoderm-derived mesenchymal stem cells (DE-MSCs) meeting the mesenchymal stem cell standard (ISCT standard, 2006) were obtained.
The mesenchymal stem cells from the definitive endoderm cells prepared in example 3 are detected by the following method:
qPCR was measured using a fluorescent quantitative PCR detector (model No. Sammerfei QuantStudio 7), total mRNA was extracted using RNAioso-plus (TAKARA, cat # 108-95-2), and reverse transcription from mRNA to cDNA was performed using a high capacity cDNA reverse transcription kit (Applied Biosystems, cat # 4368813). Real-time quantitative PCR was performed using SYBR Premix Ex Taq (TAKARA, cat # RR 420) and Quantstudio-7 system (Applied Biosystems). The relative amount of amplified nucleotide fragments was calculated by the 2^ (- Δ Ct) method. Expression levels were normalized to the housekeeping gene TBP and compared to undifferentiated embryonic stem cells.
Flow cytometry was measured using a flow cytometer (model: BD C6), and cells were harvested by trypLE (37 ℃,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.
For the differentiation protocol provided in this example, please refer to FIG. 12, and for the assay results, please refer to FIGS. 13-15.
As can be seen in figure 12, this example provides a protocol for activating the Wnt pathway and inhibiting the TGF pathway in days 3-5, and figure 13 represents a qPCR assay to test the effect of CHIR99021, SB431542, XAV939, and combinations thereof on CD44, CD73, CD105mRNA expression levels on day 15. As can be seen from fig. 14, after 2-3 passages, the cell morphology was classified as uniform at day 24, a fusiform cell morphology, suggesting the formation of mesenchymal stem cells.
As can be seen from FIG. 15, DE-MSCs (FBS) indicates DE treatment in the presence of FBS only, DE-MSCs (CHIR) indicates DE treatment with CHIR99021 in the presence of FBS for an additional 2 days; DE-MSCs (CHIR/SB) means that DE was treated with CHIR99021 and SB431542 in the presence of FBS for an additional 2 days. The mesenchymal stem cell positive markers are positive CD44, CD73, CD105 and PDGFR beta at the 24 th day, and the mesenchymal stem cell negative marker is negative CD 45.
In conjunction with fig. 13-15, it can be seen that, following the derivation of definitive endoderm cells, continued activation of the Wnt pathway and inhibition of the TGF pathway contributes to the generation of definitive endoderm cell-derived mesenchymal stem cells, and these mesenchymal stem cells meet the requirements of the international association for stem cells (ISCT standard, 2006) on the mesenchymal stem cell criteria.
Example 4
This example provides a method for differentiating mesenchymal stem cells derived from Definitive Endoderm (DE), which can also be obtained via definitive endoderm via human embryonic stem cell line H1 and human induced pluripotent stem cell line NL-1.
The concrete implementation is as follows: culturing the human embryonic stem cell line H1 and the human induced pluripotent stem cell line NL-1 in an E8 culture medium, replacing the fresh culture medium every day, and carrying out passage when the cell density reaches 70-80%. DPBS-EDTA (commercially available from Cell system, cat. No. 4Z 0-610) was first washed 2 times, then incubated at room temperature for 5 minutes, DPBS-EDTA aspirated 3 times, and a well plate of Matrigel containing 5. Mu.M ROCK inhibitor was added. After the cells are attached to the wall for 1 day, the E8 culture medium is changed for continuous culture. When the cells grew to 30-60% differentiation was initiated, the first day a first differentiation medium containing CHIR99021-HCl 5. Mu.M and Activin A100ng/ml was added. On days 2 to 3 of differentiation, CHIR99021 was removed, but Activin a was maintained to induce the formation of definitive endoderm cells. On day 3, definitive endoderm cells were purified using flow cytometric sorting techniques. Culturing definitive endoderm cells in MSC production medium (days 3-5); changing a fresh MSC generation culture medium from the 5 th day to the 15 th day of differentiation, changing the culture medium once every two days, and inducing to generate a cell population containing mesenchymal stem cells; after 15 days, passage with trypsin-based digests, typically 2-3 passages, definitive endoderm cell-derived mesenchymal stem cells (DE-MSCs) meeting mesenchymal stem cell standards (ISCT standards, 2006) were obtained.
Flow cytometry was measured using a flow cytometer (commercially available from BD, model BD C6), cells were harvested by TrypLE (37 ℃,5 min) and neutralized with 5% fbs. After washing with DPBS with 1% bsa, the antibodies were incubated, after washing, the cells were resuspended in DPBS and analyzed by BD C6.
FIG. 16 represents the cell morphology of definitive endoderm cell-derived mesenchymal stem cells at day 24 via NL-1 and H1, both in spindle shape, showing mesenchymal stem cell characteristics.
Figure 17 represents that mesenchymal stem cells derived from definitive endoderm cells were all positive for mesenchymal stem cell biomarkers (CD 44 and CD 105) at day 24 via NL-1 and H1.
The results of FIGS. 16 and 17 show that mesenchymal stem cells can be obtained via definitive endoderm as well via human embryonic stem cell line H1 and human induced pluripotent stem cell line NL-1.
Example 5
Definitive endoderm-derived mesenchymal stem cells have the potential to treat ulcerative colitis.
Differentiation of definitive endoderm-derived mesenchymal stem cells was performed as above, and animal experiments were performed after obtaining DE-MSCs (FBS, FBS CHIR and FBS CHIR/SB conditions) to examine the potential of DE-MSCs to treat ulcerative colitis. The specific method comprises the following steps: mice were given 2% Dextran Sodium Sulfate (DSS) (commercially available from MP biomedicalal, molecular weight 36,000-50,000) in drinking water for 6 days to construct a colitis model. Each mouse in the treatment group was injected intraperitoneally with 5X 10 injections on days 3 and 4 after the start of DSS 6 DE-MSCs (in FBS, FBS CHIR and FBS CHIR/SB conditions) or human UC-MSC or an equal volume of PBS. The body weight of the mice was measured daily from day 0 to day 14. On day 14, by CO 2 These mice were euthanized by asphyxiation. After necropsy, the colon was dissected from each mouse and its length was measured.
As can be seen from fig. 18, the body weight of the mice injected with the mesenchymal stem cell group recovered faster compared to the mice not injected with mesenchymal stem cells, indicating that the colitis disease condition of the mice recovered faster. As can be seen from fig. 19 and 20, the average colon length of the mice injected with the mesenchymal stem cell group was significantly longer than that of the mice not injected with the mesenchymal stem cells.
In summary, the differentiation method of mesenchymal stem cells derived from definitive endoderm cells provided by the present application induces differentiation of human pluripotent stem cells into definitive endoderm cells (DE) and mesenchymal stem cells (DE-MSCs) specific to definitive endoderm cells (DE) using a well-defined method. More importantly, the use of WNT pathway activators, WNT pathway inhibitors and TGF pathway inhibitors in combination with short-term processing of definitive endoderm cells (DE) will help these cells to obtain midgut cell, hindgut cell and foregut cell fates; the mesenchymal stem cells derived from the definitive endoderm of the above human pluripotent stem cells are collectively referred to as DE-MSCs. DE-MSCs obtained by the way of cell fate of midgut/hindgut are similar to MSCs derived from human small intestine and large intestine, and are suitable for treating digestive tract related diseases represented by colitis and Crohn's disease. The application provides an in vitro differentiation method for efficiently preparing definitive endoderm-derived mesenchymal stem cells derived from human pluripotent stem cells. And the DE-MSCs can secrete anti-inflammatory factors under the condition of in vitro stimulation, and further, the DE-MSCs injected by the abdominal cavity have the potential of treating the colitis of the mice.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for differentiating mesenchymal stem cells derived from definitive endoderm cells, comprising:
a first differentiation stage: inducing differentiation of human pluripotent stem cells into mesendoderm cells by culturing for 1-2 days in a first differentiation medium containing a WNT pathway activator and Activin A;
a second differentiation stage: inducing the formation of definitive endoderm cells (DE) by culturing for 2-8 days in a second differentiation medium without WNT pathway activator and containing Activin A;
a third differentiation stage: inducing said definitive endoderm cells to obtain fates of midgut cells, hindgut cells, and foregut cells by culturing for 2-4 days in MSC production medium;
and a fourth differentiation stage: continuously culturing in the MSC generating culture medium for 8-14 days to induce and generate a cell population containing the mesenchymal stem cells;
a fifth separation stage: digesting and passaging for 2-3 times to obtain mesenchymal stem cells (DE-MSCs) from the definitive endoderm cells.
2. The method of differentiating definitive endoderm cell-derived mesenchymal stem cells according to claim 1, further comprising, after the end of the second differentiation stage, purifying the definitive endoderm cells (DE) using flow cytometric sorting techniques;
preferably, purifying the definitive endoderm cells (DE) comprises: the cells were harvested by cell digest and neutralized with neutralization solution, washed, resuspended and purified by flow cytometry.
3. The method of differentiating definitive endoderm cell-derived mesenchymal stem cells according to claim 1 or 2, wherein a cell signaling pathway modulator is added to the MSC production medium at a third differentiation stage,
preferably, the cell signaling pathway modulator comprises at least one of a WNT pathway activator, a WNT pathway inhibitor, and a TGF pathway inhibitor.
4. The method of differentiating definitive endoderm cell-derived mesenchymal stem cells according to claim 3, wherein the WNT pathway activator comprises at least one of CHIR99021, CHIR99021-HCl, wnt3a ligand, wnt5a ligand, and BIO; the WNT pathway inhibitor comprises at least one of XAV939, IWP2, and IWR-1; the TGF pathway inhibitor comprises one of SB431542, repsox, SD-208, GW788388, A-77-01 and A-83-01;
preferably, the WNT pathway activator is CHIR99021 at a treatment concentration of 1-5 μ Μ;
preferably, the WNT pathway inhibitor is XAV939 at a treatment concentration of 1-10 μ Μ;
preferably, the TGF pathway inhibitor is SB431542 at a treatment concentration of 1-10. Mu.M.
5. The method of differentiation of definitive endoderm cell-derived mesenchymal stem cells according to claim 1, wherein the first differentiation medium is obtained by adding a basal component to the WNT pathway activator and Activin a, and the second differentiation medium is obtained by adding a basal component to the Activin a;
preferably, the WNT pathway activator is CHIR99021-HCl with the treatment concentration of 1-5 mu M, and the addition amount of Activin A is 10-100ng/ml;
preferably, the basic components include DMEM/F12, L-ascorbic acid-2-magnesium phosphate 60-70mg/L, sodium selenite 10-15 μ g/L, transferrin 8-12 μ g/ml, chemical defined lipid concentrate 1 × and streptomycin 1%;
preferably, the serum-free MSC production medium comprises alpha MEM, L-ascorbic acid-2-magnesium phosphate 60-70mg/L, sodium selenite 10-15. Mu.g/L, insulin 8-12. Mu.g/ml, FGF 2-110 ng/ml, B-27 TM Supplement 1×、GlutaMAX TM Supplement 1 ×, NEAA 1 × and streptomycin 1%; or,
the components of the MSC production medium containing serum comprise alpha MEM, FBS 10% -20%, L-ascorbic acid-2-magnesium phosphate 60-70mg/L, sodium selenite 10-15 μ g/L, insulin 8-12 μ g/ml, FGF 2-110 ng/ml, B-27 TM Supplement 1×、GlutaMAX TM Supplement 1X, NEAA 1X and streptomycin 1%.
6. The method of differentiating definitive endoderm cell-derived mesenchymal stem cells according to claim 1, wherein the human pluripotent stem cells include at least one of human embryonic stem cells and human induced pluripotent stem cells;
preferably, the human embryonic stem cells include human embryonic stem cells H1 or human embryonic stem cells H9;
preferably, the human induced pluripotent stem cells comprise human induced pluripotent stem cells NL-1;
preferably, the human pluripotent stem cells are differentiated at a cell density of 30 to 60%.
7. A definitive endoderm cell-derived mesenchymal stem cell obtained by differentiation using the method for differentiating a definitive endoderm cell-derived mesenchymal stem cell according to any one of claims 1 to 6.
8. Use of definitive endoderm cell-derived mesenchymal stem cells of claim 7 in the preparation of a medicament for the treatment of a gut-related disease.
9. A mesenchymal stem cell-derived mesenchymal stem cell according to claim 7, which is obtained by in vitro differentiation of the definitive endoderm cell-derived mesenchymal stem cell;
preferably, the differentiated cells of the mesenchymal stem cells include at least one of adipocytes, chondrocytes, and osteoblasts.
10. A kit for inducing differentiation of human pluripotent stem cells into mesenchyme stem cells derived from definitive endoderm cells, which comprises a reagent for carrying out the method for differentiating definitive endoderm cell-derived mesenchyme stem cells according to any one of claims 1 to 6.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117721077A (en) * | 2024-02-08 | 2024-03-19 | 广东乾晖生物科技有限公司 | Compositions, complete media, methods and uses for inducing differentiation of iPSC to MSC |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120052587A (en) * | 2010-11-16 | 2012-05-24 | 고려대학교 산학협력단 | Method for the transdifferentiation of mesenchymal stem cells into endodermal cell |
| US20130273013A1 (en) * | 2010-12-15 | 2013-10-17 | Michel Revel | Insulin producing cells derived from pluripotent stem cells |
| CN111417716A (en) * | 2017-11-30 | 2020-07-14 | 公立大学法人横滨市立大学 | Construction of three-dimensional organs from pluripotent stem cells |
| CN112655702A (en) * | 2020-12-31 | 2021-04-16 | 青岛奥克生物开发有限公司 | Solution for umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cell preparation, preparation method and application |
| CN112831461A (en) * | 2021-02-26 | 2021-05-25 | 澳门大学 | Method and medicine for inducing stem cells to differentiate into mesoderm lineages or trophoblast lineages |
| WO2024063533A1 (en) * | 2022-09-21 | 2024-03-28 | 한국생명공학연구원 | High-performance mature intestinal organoid regenerative therapeutic agent derived from pluripotent stem cells |
-
2022
- 2022-09-02 CN CN202211072699.3A patent/CN115232788A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120052587A (en) * | 2010-11-16 | 2012-05-24 | 고려대학교 산학협력단 | Method for the transdifferentiation of mesenchymal stem cells into endodermal cell |
| US20130273013A1 (en) * | 2010-12-15 | 2013-10-17 | Michel Revel | Insulin producing cells derived from pluripotent stem cells |
| CN111417716A (en) * | 2017-11-30 | 2020-07-14 | 公立大学法人横滨市立大学 | Construction of three-dimensional organs from pluripotent stem cells |
| CN112655702A (en) * | 2020-12-31 | 2021-04-16 | 青岛奥克生物开发有限公司 | Solution for umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cell preparation, preparation method and application |
| CN112831461A (en) * | 2021-02-26 | 2021-05-25 | 澳门大学 | Method and medicine for inducing stem cells to differentiate into mesoderm lineages or trophoblast lineages |
| WO2024063533A1 (en) * | 2022-09-21 | 2024-03-28 | 한국생명공학연구원 | High-performance mature intestinal organoid regenerative therapeutic agent derived from pluripotent stem cells |
Non-Patent Citations (2)
| Title |
|---|
| ZHANG, YM等: "Definitive endodermal cells supply an in vitro souce of mesenchymal stem/stromal cells", 《COMMUNICATIONS BIOLOGY》, vol. 6, no. 1, 12 May 2023 (2023-05-12), pages 1 - 11 * |
| 李静等: "人脂肪间充质干细胞向限定性内胚层细胞分化体系的优化", 《基础医学与临床》, vol. 39, no. 07, 5 July 2019 (2019-07-05), pages 954 - 960 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117721077A (en) * | 2024-02-08 | 2024-03-19 | 广东乾晖生物科技有限公司 | Compositions, complete media, methods and uses for inducing differentiation of iPSC to MSC |
| CN117721077B (en) * | 2024-02-08 | 2024-05-31 | 广东乾晖生物科技有限公司 | Compositions, complete media, methods and uses for inducing differentiation of iPSC to MSC |
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