CN111471645A - Method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using small molecules - Google Patents
Method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using small molecules Download PDFInfo
- Publication number
- CN111471645A CN111471645A CN202010322188.7A CN202010322188A CN111471645A CN 111471645 A CN111471645 A CN 111471645A CN 202010322188 A CN202010322188 A CN 202010322188A CN 111471645 A CN111471645 A CN 111471645A
- Authority
- CN
- China
- Prior art keywords
- cells
- liver
- inducing
- induction
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using small molecules, which induces the definitive endoderm cells; induction of liver progenitor cells from DE cells; and (4) obtaining liver mature cells by induction. The invention has the advantages that the hAD-MSCs are obtained and the in vitro culture conditions are mature, can be derived from the adipose tissues of patients without ethical obstacles to avoid the immunological rejection of allogeneic cells, and have no immunogenicity and high cell proliferation activity; the iHeps with the morphology, gene expression and function of the liver cells can be obtained only in about 15 days, the induction process is rapid and efficient, and the iHeps can become a stable source for liver cell transplantation in a clinical treatment scheme.
Description
Technical Field
The invention belongs to the technical field of stem cells and regenerative medicine, and relates to a method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using chemical micromolecules.
Background
The use of O L T is an effective means for treating end-stage liver diseases, however, the use of O L T is limited by donor deficiency, side effects of immunosuppressive agents and ethical issues, liver cell transplantation and artificial liver support become effective methods for restoring liver function, increasing patient survival rates, but both methods are also limited due to limited cell sources and equipment deficiencies, liver cell transplantation contributes to the restored function of liver with functional failure.
Disclosure of Invention
The invention aims to provide a method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using small molecules, and solves the problem that (1) a primary hepatocyte source (2) in vitro induction of functional hepatocytes is long in the prior art. The invention has the beneficial effects that: (1) the hAD-MSCs are obtained and cultured in vitro under mature conditions, can be derived from the adipose tissues of patients without ethical obstacles to avoid immunological rejection of allogeneic cells, and have no immunogenicity and high cell proliferation activity; (2) the iHeps with the morphology, gene expression and function of the liver cells can be obtained only in about 15 days, the induction process is rapid and efficient, and the iHeps can become a stable source for liver cell transplantation in a clinical treatment scheme.
The technical scheme adopted by the invention is carried out according to the following steps:
1) inducing definitive endoderm cells;
2) induction of liver progenitor cells from DE cells;
3) and (4) obtaining liver mature cells by induction.
Further, the method of inducing definitive endoderm cells is as follows:
(1) selecting fourth generation hAD-MSCs as seed cells, and digesting for later use;
(2) inoculating the cells of the experimental group and the cells of the control group into a pore plate, and adding an hAD-MSCs culture medium for culture;
(3) discarding the culture medium, washing with PBS, adding DMSO-containing serum F12-DMEM culture medium, and inducing;
(4) discarding the induction culture medium containing DMSO, washing with PBS, adding serum F12-DMEM culture medium containing CHIR99021, and inducing;
(5) discarding a serum-containing F12-DMEM culture medium containing MCHIR99021, washing with PBS once, adding an F12-DMEM culture medium, and inducing;
(6) cell samples were collected and tested for expression of the marker genes Foxa2, Sox17 by a variety of molecular means.
Further, the method for obtaining liver progenitor cells from DE cells by induction is as follows:
(1) DE cells obtained by induction of fourth generation hAD-MSCs are used as seed cells;
(2) discarding the F12-DMEM medium, washing with PBS, and adding a serum-free F12-DMEM medium containing SB431542, sodium butyrate and DMSO;
(3) changing the liquid for one time every day;
(4) cell samples were collected and tested for expression of the Foxa2, a L B, HNF4 α marker gene by various methods.
Further, the method for obtaining liver mature cells by induction is as follows:
(1) after the liver progenitor cells are obtained by induction, discarding serum-free F12-DMEM medium containing SB431542 and sodium butyrate, washing once with PBS, and adding serum-free F12-DMEM medium containing FH1, FPH1, SB431542, dexmethasone and hydrocortisone;
(2) changing the liquid once a day, and inducing;
(3) cell samples were collected and tested for AFP, A L B, A1AT marker gene expression by various methods.
Drawings
FIG. 1 is a morphogram of cells after induction according to the invention;
FIG. 2 is a graph showing the result of immunofluorescence staining of liver cell A L B after induction according to the present invention;
FIG. 3 is a graph showing the result of glycogen staining of liver cells after induction according to the present invention.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
1. Induction of Definitive Endoderm (DE) cells
(1) Selecting fourth-generation hAD-MSCs human adipose-derived mesenchymal stem cells as seed cells, and digesting for later use;
(2) the cells of the experimental group and the control group are treated with 5 × 105Inoculating the cells/hole density into a 6-hole plate, adding an hAD-MSCs culture medium to 2 ml/hole, and culturing for 24 h;
(3) the medium was discarded, washed once with PBS, and added with 2% serum F12-DMEM modified Eagle medium containing 0.5% DMSO: the nutrient mixture F-12 culture medium, 2 ml/hole, induces for 24 h;
(4) the induction medium containing 0.5% DMSO was discarded, washed once with PBS,adding CHIR99021(3 μ M) (C)22H18Cl2N8) 2% serum F12-DMEM culture medium, 2 ml/hole, inducing for 24 h;
(5) the 2% serum-containing F12-DMEM medium containing 3. mu.MCHIR 99021 was discarded, washed once with PBS, added to F12-DMEM medium at 2 ml/well, and induced for 24 hours.
(6) After 3 days of co-culture, cell samples were collected and the expression of marker genes Foxa2, Sox17, etc. was examined by various molecular means.
2. Induction of liver Progenitor (liver cells) cells from DE cells
(1) DE cells obtained by induction of fourth generation hAD-MSCs are used as seed cells;
(2) discard F12-DMEM medium, wash once with PBS, add SB431542 (0.5. mu.M) (C)22H16N4O3) Serum free F12-DMEM medium, 2 ml/well, of sodiumbutyrate (250nM) (sodium butyrate) and DMSO (0.5% of the total volume) (dimethyl sulfoxide);
(3) changing the liquid once a day, and inducing for 7 days;
(4) after 7 days of co-culture, cell samples were collected and tested for expression of marker genes such as Foxa2 and A L B, HNF4 α by various methods.
3. Inducing to obtain liver mature cells (Hepatocyte-like cells)
(1) After obtaining liver progenitor cells by induction, SB431542 (C) was discarded22H16N4O3) And sodium
Serum-free F12-DMEM Du's modified Eagle Medium for butyrate (sodium butyrate): the nutrient mixture F-12, washed once with PBS and supplemented with FH1 (15. mu.M) (C)17H18N2O2)，FPH1(15μM)(C16H15ClF2N2O3S)，SB431542(0.5μM)(C22H16N4O3) Serum-free F12-DMEM medium, 2 ml/well, of dexamethasone (100nM) (dexamethasone) and hydrocortisone (10. mu.M) (hydrocortisone);
(2) changing the liquid once a day, and inducing for 6 days;
(3) after 6 days of co-culture, cell samples were collected and tested for expression of marker genes such as AFP, A L B, A1AT by different methods.
FIG. 1 is a diagram showing morphology of a liver cell (SM-iHep) induced by a Small Molecule (SM) under a phase contrast microscope, wherein the morphology of the liver cell is changed by 10 times and the scale bar is 200 μm, the liver cell is in a spindle shape at the initial stage of culture during induction, adherent clones are formed, and the decrease of the adherent ability gradually becomes oval after 2 weeks (SMd17, day 17 after the induction of the Small molecule). fig. 2 is a diagram showing immunofluorescence staining results of the liver cell A L B after the induction of the present invention, the left is a diagram showing the expression level of a liver cell function marker Albumin (Albumin, A L B), the middle is 4', 6-diamidino-2-phenylindole (DAPI) stained cell nucleus, the right is synthesized by the two, fig. 3 is a diagram showing glycogen staining results of the liver cell after the induction of the present invention, and the Periodic Acid Schiff's (PAS) staining method shows that the liver cell is purplish red or red liver glycogen, which shows that the induced liver cell can be synthesized into glycogen storage 3502, and serves as a control line of a normal human cell group L02.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
Claims (4)
1. The method for inducing the human adipose-derived mesenchymal stem cells to be differentiated into liver-like cells by using small molecules is characterized by comprising the following steps of:
1) inducing definitive endoderm cells;
2) induction of liver progenitor cells from DE cells;
3) and (4) obtaining liver mature cells by induction.
2. The method for inducing the differentiation of human adipose-derived mesenchymal stem cells into liver-like cells by using the small molecule according to claim 1, which is characterized in that: the method for inducing definitive endoderm cells is as follows:
(1) selecting fourth generation hAD-MSCs as seed cells, and digesting for later use;
(2) inoculating the cells of the experimental group and the cells of the control group into a pore plate, and adding an hAD-MSCs culture medium for culture;
(3) discarding the culture medium, washing with PBS, adding DMSO-containing serum F12-DMEM culture medium, and inducing;
(4) discarding the induction culture medium containing DMSO, washing with PBS, adding serum F12-DMEM culture medium containing CHIR99021, and inducing;
(5) discarding a serum-containing F12-DMEM culture medium containing CHIR99021, washing with PBS once, adding F12-DMEM culture medium, and inducing;
(6) cell samples were collected and tested for expression of the marker genes Foxa2, Sox17 by a variety of molecular means.
3. The method for inducing the differentiation of human adipose-derived mesenchymal stem cells into liver-like cells by using the small molecule according to claim 1, which is characterized in that: the method for obtaining liver progenitor cells from DE cells by induction comprises the following steps:
(1) DE cells obtained by induction of fourth generation hAD-MSCs are used as seed cells;
(2) discarding F12-DMEM medium, washing with PBS, and adding serum-free F12-DMEM medium containing SB431542, sodimubutyrate and DMSO;
(3) changing the liquid for one time every day;
(4) cell samples were collected and tested for expression of the Foxa2, a L B, HNF4 α marker gene by various methods.
4. The method for inducing the differentiation of human adipose-derived mesenchymal stem cells into liver-like cells by using the small molecule according to claim 1, which is characterized in that: the method for obtaining the liver mature cells through induction comprises the following steps:
(1) after obtaining the liver progenitor cells by induction, discarding serum-free F12-DMEM medium containing SB431542 and sodiumbutylate, washing with PBS, and adding serum-free F12-DMEM medium containing FH1, FPH1, SB431542, dexamethasone and hydrocortisone;
(2) changing the liquid once a day, and inducing;
(3) cell samples were collected and tested for AFP, A L B, A1AT marker gene expression by various methods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010322188.7A CN111471645A (en) | 2020-04-22 | 2020-04-22 | Method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using small molecules |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010322188.7A CN111471645A (en) | 2020-04-22 | 2020-04-22 | Method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using small molecules |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111471645A true CN111471645A (en) | 2020-07-31 |
Family
ID=71760485
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010322188.7A Pending CN111471645A (en) | 2020-04-22 | 2020-04-22 | Method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells by using small molecules |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111471645A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168065A (en) * | 2011-02-17 | 2011-08-31 | 暨南大学 | Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof |
| WO2018036119A1 (en) * | 2015-11-19 | 2018-03-01 | 中国人民解放军第二军医大学 | Method for in-vitro induction of ductal metaplasia of primary hepatocyte and for long-term cultivation, proliferation and differentiation thereof, and application thereof |
| US20180055887A1 (en) * | 2016-08-23 | 2018-03-01 | Academia Sinica | Method for preparing induced mesenchymal stem cells and improving mesenchymal stem cell's characters and its applications |
| CN108486037A (en) * | 2018-02-12 | 2018-09-04 | 中山大学附属第三医院 | A method of being divided into liver cell using micromolecular compound induction human pluripotent stem cells |
| CN109082401A (en) * | 2018-07-31 | 2018-12-25 | 南昌大学 | A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional hepatocytes |
-
2020
- 2020-04-22 CN CN202010322188.7A patent/CN111471645A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168065A (en) * | 2011-02-17 | 2011-08-31 | 暨南大学 | Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof |
| WO2018036119A1 (en) * | 2015-11-19 | 2018-03-01 | 中国人民解放军第二军医大学 | Method for in-vitro induction of ductal metaplasia of primary hepatocyte and for long-term cultivation, proliferation and differentiation thereof, and application thereof |
| US20180055887A1 (en) * | 2016-08-23 | 2018-03-01 | Academia Sinica | Method for preparing induced mesenchymal stem cells and improving mesenchymal stem cell's characters and its applications |
| CN108486037A (en) * | 2018-02-12 | 2018-09-04 | 中山大学附属第三医院 | A method of being divided into liver cell using micromolecular compound induction human pluripotent stem cells |
| CN109082401A (en) * | 2018-07-31 | 2018-12-25 | 南昌大学 | A kind of amnioic epithelium stem cell is induced to differentiate into the method and its application of functional hepatocytes |
Non-Patent Citations (1)
| Title |
|---|
| 余跃等: "《干细胞基础与临床》", 29 February 2008, 中国科学技术大学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hu et al. | In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration | |
| Kang et al. | Differentiating characterization of human umbilical cord blood‐derived mesenchymal stem cells in vitro | |
| Yin et al. | Human umbilical cord mesenchymal stem cells ameliorate liver fibrosis in vitro and in vivo: from biological characteristics to therapeutic mechanisms | |
| Wang et al. | Cell-to-cell contact induces mesenchymal stem cell to differentiate into cardiomyocyte and smooth muscle cell | |
| Yokoo et al. | Human corneal endothelial cell precursors isolated by sphere-forming assay | |
| CN112111444B (en) | Method for reprogramming umbilical cord mesenchymal stem cells into liver cells and prepared liver organoid | |
| US20060110825A1 (en) | Process for the preparation of stem cells from human muscle tissue and adipose tissue, and stem cell obtainable by this process | |
| WO2015180636A1 (en) | Specific medium for long-term maintenance and proliferation subculture of human hepatocytes and culture method | |
| Pimton et al. | Hypoxia enhances differentiation of mouse embryonic stem cells into definitive endoderm and distal lung cells | |
| Xu et al. | Three-dimensional culture promotes the differentiation of human dental pulp mesenchymal stem cells into insulin-producing cells for improving the diabetes therapy | |
| WO2011101834A1 (en) | A method for obtaining mesenchymal stem cells, media, methods and composition thereof | |
| JP2005521405A (en) | Dedifferentiated programmable stem cells originating from monocytes and their production and use | |
| CN109082401B (en) | Method for inducing and differentiating amniotic epithelial stem cells into functional liver cells and application thereof | |
| CN109706115B (en) | Construction method of mouse bone marrow mesenchymal stem cell line | |
| TW200902716A (en) | Complexes of hyaluronans, other matrix components, hormones and growth factors for maintenance, expansion and differentiation of hepatic cells | |
| WO2007102162A2 (en) | Regulating stem cells | |
| CN109517783B (en) | Full-small-molecule culture medium for liver organoid culture and application thereof | |
| Kao et al. | Comparisons of differentiation potential in human mesenchymal stem cells from Wharton’s jelly, bone marrow, and pancreatic tissues | |
| Saito et al. | Current status of hepatocyte-like cell therapy from stem cells | |
| WO2011016485A1 (en) | METHOD FOR INDUCING DIFFERENTIATION OF iPS CELLS INTO HEPATIC PARENCHYMAL CELLS | |
| Raju et al. | The road to regenerative liver therapies: the triumphs, trials and tribulations | |
| CN110951686A (en) | Hematopoietic stem cell in-vitro amplification culture system and method | |
| CN108048390B (en) | Method for preparing vascular endothelial cells and special kit thereof | |
| US9404084B2 (en) | Regulating stem cells | |
| RU2510276C1 (en) | Method for preparing cells for replacement cell therapy of hepatic pathology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200731 |
|
| RJ01 | Rejection of invention patent application after publication |