CN104388383A - Long-term in-vitro culture and directional differentiation system and method for liver stem cell - Google Patents
Long-term in-vitro culture and directional differentiation system and method for liver stem cell Download PDFInfo
- Publication number
- CN104388383A CN104388383A CN201410635815.7A CN201410635815A CN104388383A CN 104388383 A CN104388383 A CN 104388383A CN 201410635815 A CN201410635815 A CN 201410635815A CN 104388383 A CN104388383 A CN 104388383A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- stem cells
- liver stem
- liver
- cell culture
- 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.)
- Granted
Links
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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/12—Hepatocyte growth factor [HGF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/237—Oncostatin M [OSM]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/73—Hydrolases (EC 3.)
- C12N2501/734—Proteases (EC 3.4.)
Abstract
The invention relates to the technical field of biomedical engineering and particularly relates to a long-term in-vitro culture and directional differentiation system and method for a liver stem cell. The long-term in-vitro culture and directional differentiation system comprises an amplification culture medium with specific chemical components, and a differential medium with specific chemical components, wherein the amplification culture medium is used for carrying out in-vitro culture of a mouse or human liver stem cell, and the differential medium is used for carrying out induced differentiation on the mouse or human liver stem cell to form a matured liver cell. By using the long-term in-vitro culture and directional differentiation system and method, a selectively-amplified liver stem cell in mother cells of the liver can be obtained from a mouse embryonic liver tissue or through human multipotential stem cell differentiation, the liver stem cell can be cultured for more than 20 generations under such a condition, and the stable molecular phenotype of the liver stem cell is maintained. By using the long-term in-vitro culture and directional differentiation system and method, the cultured mouse or human liver stem cell can be further subjected to induced differentiation to form the matured liver cell with functions of secreting albumin, metabolizing urea and the like.
Description
Technical field
The present invention relates to bio-pharmaceutical engineer technology domain, the long-term in vitro being specifically related to a kind of liver stem cells is cultivated and directed differentiation system and way, comprises the amplification culture medium that a kind of Chemical Composition is clear and definite, for the vitro culture of mouse or people's liver stem cells; And the division culture medium that a kind of Chemical Composition is clear and definite, for mouse or people's liver stem cells are induced to differentiate into mature hepatocytes.
Background technology
China every year because the patient of hepatopathy death is more than 300,000, but only can meet the demand of 5% liver problem sufferer every year for liver transplantation.The clinical application of this treatment means seriously can be limited for the scarcity of liver transplantation.Existing clinical before and clinical study show, can replant in liver injury and rebuild liver after mature hepatocytes or liver stem cell transplantation, but be no matter that mature hepatocytes or liver stem cells exist the deficient problem in source equally.
By human pluripotent stem cells (embryonic stem cell and induced pluripotent stem cells) Differentiation Induction in vitro for the Transplanted cells that not only can be liver problem sufferer of the liver stem cells cultivated of stabilization in vitro stable cell derived can be provided, and avoiding the ethnics Problem utilized involved by tire hepatic tissue, advantage is apparent.But the directional induction of multipotential stem cell is at present all the atomization of the timing by multi-step mostly, final acquisition has cell (the Wobus AM & Boheler KREmbryonic Stem Cells:Prospects for Developmental Biology and Cell Therapy.Physiol.Rev.2005 of function, whole end differentiation; 85 (2): 635-678.) at present, still differentiated system stably cannot be maintained specific middle etap, such as tissue stem cell stage.Meanwhile, at present also without effective method human pluripotent stem cells being induced to differentiate into the liver stem cells of Absorbable organic halogens amplification.
Therefore, this area still lacks effective means amplifying liver stem cells, especially cannot realize under the culture condition of specific chemical components liver stem cells as long-term in vitro cultivate and directed differentiation.
Summary of the invention
The object of the present invention is to provide a kind of long-term in vitro of liver stem cells to cultivate and directed differentiation system, this system comprises the clear and definite amplification culture medium of a kind of Chemical Composition, for the vitro culture of mouse or people's liver stem cells; And the division culture medium that a kind of Chemical Composition is clear and definite, for mouse or people's liver stem cells are induced to differentiate into mature hepatocytes.
Another object of the present invention is to provide a kind of long-term in vitro cultivation of liver stem cells and the method for directed differentiation, human pluripotent stem cells can be induced to differentiate into liver stem cells by the method, and can maintain liver stem cells long-term stability and increase and keep directed differentiation to be the ability of mature hepatocytes.
A first aspect of the present invention, is to provide the long-term in vitro culture system of a kind of mouse or people's liver stem cells, and this system is the clear and definite amplification culture medium for cultivating mouse or people's liver stem cells of a kind of Chemical Composition, and described amplification culture medium comprises:
Liquid basal medium,
0.1%-10% (volumn concentration) Insulin-Transferrin-Sodium Selenite mixed solution,
The glycogen synthase kinase-3 beta inhibitor of 3nM-10 μM,
The transforming growth factor β receptor inhibitor of 0.1 μM-50 μMs,
The Ultrapole L of 0.1 μM-50 μMs,
The S1P of 0.1 μM-50 μMs,
The Urogastron of 0.1-100ng/mL,
With people's recombinant albumin or the bovine serum albumin of 0.1-1mg/mL.
Described liquid basal medium, be selected from MEM cell culture medium, DMEM cell culture medium, DMEM/F12 cell culture medium, IMDM cell culture medium, 199 cell culture mediums, F10 cell culture medium, F12 cell culture medium, 1640 cell culture mediums one or more.
Preferably, described amplification culture medium contains the glycogen synthase kinase-3 beta inhibitor of 0.5 μM-5 μMs;
Preferably, described amplification culture medium contains the transforming growth factor β receptor inhibitor of 1 μM-10 μMs;
Preferably, described amplification culture medium contains the Ultrapole L of 1 μM-10 μMs;
Preferably, described amplification culture medium contains the S1P of 0.1 μM-5 μMs;
Preferably, described amplification culture medium contains the Urogastron of 1-50ng/mL.
Preferably, described amplification culture medium also comprises: the vitamins C of 0.1-5mM nicotinamide and 0.1-100 μ g/mL.
Described glycogen synthase kinase-3 beta inhibitor can be CHIR99021 or other small molecules glycogen synthase kinase-3 beta inhibitors;
Described transforming growth factor β receptor inhibitor can be E-616452 or other transforming growth factor β receptor inhibitor.
A second aspect of the present invention, is to provide the directed differentiation system of a kind of mouse or people's liver stem cells, and this system is the clear and definite division culture medium that liver stem cells can be induced to differentiate into mature hepatocytes of a kind of Chemical Composition, and described division culture medium comprises:
Liquid basal medium,
0.1-100ng/mL pHGF,
0.1-100ng/mL oncostatinM,
The dexamethasone of 10nM-10 μM,
The inhibitors of gamma-secretase Compound E of 10nM-10 μM, and
The transforming growth factor β receptor inhibitor E-616452 of 0.1 μM-50 μMs.
Described liquid basal medium, be selected from MEM cell culture medium, DMEM cell culture medium, DMEM/F12 cell culture medium, IMDM cell culture medium, 199 cell culture mediums, F10 cell culture medium, F12 cell culture medium, 1640 cell culture mediums one or more.
Preferably, described division culture medium contains 5-50ng/mL pHGF;
Preferably, described division culture medium contains 5-50ng/mL oncostatinM;
Preferably, described division culture medium contains the dexamethasone of 50nM-5 μM;
Preferably, described division culture medium contains the inhibitors of gamma-secretase Compound E of 100nM-5 μM;
Preferably, described division culture medium contains the transforming growth factor β receptor inhibitor E-616452 of 1 μM-20 μMs.
A third aspect of the present invention, be to provide the long-term in vitro cultivation of a kind of mouse or people's liver stem cells and the method for directed differentiation, the method comprises the following steps:
A, utilize aforesaid amplification culture medium, selective amplification liver stem cells from liver mother (Hepatoblasts) cell, and vitro culture is carried out to liver stem cells and goes down to posterity;
B, utilize aforesaid division culture medium, the liver stem cells that steps A obtains is induced to differentiate into mature hepatocytes.
In steps A, described liver stem cells liver parent cell obtains from embryo liver in mice tissue or by human pluripotent stem cells differentiation.
Liver parent cell stem cell is obtained from embryo liver in mice tissue, for general knowledge known in this field, can see document: Weiss MC, Strick-Marchand H.Isolation and characterization of mousehepatic stem cells in vitro.Semin Liver Dis 2003; 23:313-324..
Broken up by human pluripotent stem cells and obtain liver parent cell stem cell, for general knowledge known in this field, can see document: Takayama K, Nagamoto Y, Mimura N, Tashiro K, Sakurai F, TachibanaM, Hayakawa T, et al.Long-Term Self-Renewal of Human ES/iPS-DerivedHepatoblast-like Cells on Human Laminin 111-Coated Dishes.Stem CellReports 2013; 1:322-335.
Method of the present invention, can obtain selective amplification liver stem cells liver parent cell from embryo liver in mice tissue or by human pluripotent stem cells differentiation, cultivated by liver stem cells long-term in vitro, and Induction of committed differentiation be mature hepatocytes.
A fourth aspect of the present invention, is to provide and cultivates according to the long-term in vitro of aforesaid a kind of mouse or people's liver stem cells and liver stem cells that the method for directed differentiation prepares and mature hepatocytes.
A fifth aspect of the present invention, is to provide and is preparing the application in the treatment cell of hepatic diseases, liver tissue engineering, the external drug screening of hepatic diseases according to described liver stem cells and mature hepatocytes.
Beneficial effect of the present invention: the liver stem cells amplification culture medium using Chemical Composition of the present invention clear and definite can obtain selective amplification liver stem cells liver parent cell from embryo liver in mice tissue or by human pluripotent stem cells differentiation, liver stem cells can be cultivated with this understanding more than 20 generations, and maintains stable liver stem cells molecular phenotype; Method of the present invention without the need to use the complexity of magnetic bead or airflow classification, the cell purification means of high cost, the liver stem cells purity of cultivating can reach 99%.The mouse of cultivation or people's liver stem cells can be induced to differentiate into by the hepatic stem cell differentiation substratum using Chemical Composition of the present invention clear and definite can Albumin Secretion, the mature hepatocytes of the functions such as metabolizing urea.
Accompanying drawing explanation
Fig. 1 is that the amplification cultivation based selective utilizing Chemical Composition clear and definite cultivates mice embryonic liver stem cells, after cell cultures to the 5th generation, the expression (A) of endotheliocyte and mesenchymal cell gene namely cannot be detected; And the liver stem cells purity of the EpCAM positive can reach 98.5% (B); In cell cultures to 20 generation, still can keep normal caryogram (C), and stable gene expression profile (D).
Fig. 2 is that cultured cells has the phenotype of liver stem cells, expresses the marker molecule of liver stem cells, as coexpression Albumin (ALB) and keratin 19 (K19), liver precursor marker DLK1, EpCAM and CD133, liver endogenous transcription factor HNF4 α and FoxA2, simultaneously coexpression epithelial cell molecular marked compound E-Cadherin and β-Catenin (A); Flowcytometric results showed cell expresses CD133 (99% positive rate), Ki-67 (88% positive rate), DLK1 (94% positive rate), and K19 (99%positive) (B); Immunofluorescence dyeing result shows, and the single liver stem cells of cultivation has two-way differentiation potential, can be divided into the liver cell of the ALB positive and the bile duct epithelial cell (C) of the K19 positive.Fig. 3 is the bile duct spline structure that the liver stem cells cultivated is formed in three-dimensional Matrigel, A and B is that the bile duct observed under phase microscope supports structure, and B is the enlarged view of rectangle frame in A figure; C is the bile duct that transmission electron microscope observation arrives; The bile duct spline structure that immunocytochemical stain display is formed expresses the marker molecule (D, E, F) of bile duct.
The liver stem cells that Fig. 4 cultivator pluripotent stem cell differentiation obtains, cultured cells expresses the marker molecule of liver stem cells, as coexpression CD54/DLK1 (A), AFP/HNF4 (B), EpCAM/HNF4 (C), pan-CK/FoxA2 (D), E-cad/Ki-67 (E); The positive rate of flowcytometric results showed cell Ki-67 can reach 99%; The positive rate of EpCAM can reach 89% (F).
Fig. 5 is hepatic stem cell differentiation is mature hepatocytes, through the vitro culture of two weeks under induction condition, liver stem cells a high proportion ofly can be divided into the mature hepatocytes (A, B and C, B is the enlarged view of rectangle frame in A figure) of expressing and also can secrete ALB; Cell after differentiation has the ability (D) of picked-up acetylated low density lipoprotein, can take in fluorescein-labeled cholic acid and cholic acid is secreted into iuntercellular bile duct (E) simultaneously; Cell after differentiation has the ability (F) of urea synthesis.
Embodiment
Below in conjunction with drawings and Examples of the present invention, enforcement of the present invention is elaborated; following examples implement under premised on technical solution of the present invention; give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1: amplification mice embryonic liver stem cells
Get the mice embryonic of conceived 12.5 days, take out fetal liver, after Accutase enzyme (Invitrogen company) digestion, single cell suspension is inoculated in 6 well culture plates that 0.5%Matrigel matrigel (BD Bioscience company) or 20 μ g/ml Laminin (Invitrogen company) wrap quilt, substratum is DMEM/F12 (Invitrogen company), comprise Insulin-Transferrin-Sodium Selenite mixed solution (Invitrogen company), 3 μMs of CHIR99021 (Tocris company), 2 μMs of E-616452 (Sigma-Aldrich company), the Ultrapole L (Sigma-Aldrich company) of 5 μMs, the S1P (Sigma-Aldrich company) of 0.5 μM, the Urogastron (R & DSystems company) of 20ng/mL and people's recombinant albumin (R & D Systems company) of 50 μ g/mL.Secondary Culture is carried out with Accutase enzyme when Growth of Cells to 70% converges.
This culture condition can optionally increase embryonic hepatic stem cells, as shown in Figure 1, blood vessel endothelium and fibroblastic existence (Figure 1A) no longer can be detected after cell cultures five generation, and the purity of liver stem cells can reach more than 95% (Figure 1B).Liver stem cells with this understanding can continuous passage more than 20 generations, and to keep stable (Fig. 1 D) of cell caryogram (Fig. 1 C) and gene expression profile.
At the marker molecule (Fig. 2) of the cell expressing liver stem cells of this CMC model, as coexpression Albumin (ALB) and keratin 19 (K19), liver precursor marker DLK1, EpCAM and CD133, liver endogenous transcription factor HNF4 α and FoxA2, simultaneously coexpression epithelial cell molecular marked compound E-Cadherin and β-Catenin (Fig. 2 A).Flowcytometric results showed cell expresses CD133 (99% positive rate), Ki-67 (88% positive rate), DLK1 (94% positive rate), and K19 (99%positive) (Fig. 2 B).
Immunofluorescence dyeing result shows, and the single liver stem cells of cultivation has two-way differentiation potential, can be divided into the liver cell of the ALB positive and the bile duct epithelial cell (Fig. 2 C) of the K19 positive.The liver stem cells cultivated can form bile duct spline structure (Fig. 3 A, B) in three-dimensional Matrigel.The inner cavity surface of electron microscope observation display bile duct cell has a large amount of microvilluss (Fig. 3 C); And the bile duct spline structure formed expresses the marker molecule of bile duct, as (Fig. 3 D, E, F) such as K19, Integrin α 6, Integrin β 4 and Aqu1.
Above result show liver stem cells culture system that the present invention sets up can from embryo liver in mice optionally amplifying liver stem cells, and the self steady in a long-term of liver stem cells can be maintained in vitro.
Embodiment 2: the liver stem cells that the differentiation of amplification human pluripotent stem cells obtains
Human pluripotent stem cells H1 and Hues9 cell (Wicell company) are incubated at 6 well culture plates of 0.5% matrigel bag quilt, substratum is for being DMEM/F12, comprise 1%N2 (Invitrogen company), 1%B27 (Invitrogen company) and 20ng/ml bFGF (Invitrogen company).Growth of Cells to 50% replaces medium to DMEM/F12 when merging, and it is endoderm cell that B27 (without Regular Insulin), 100ng/ml Activin A (R & DSystems company) cultivates four days Cell differentiation inducing activities; Then replace medium to DMEM/F12, comprise 1%N2,1%B27,5 μMs of SB431542 (Sigma-Aldrich company) and 10ng/ml BMP4 (Invitrogen company) cultivate 2 days inducing cells and enter a part and turn to veutro anterior intestine; Then replace medium to DMEM/F12, comprise 1%N2, it is liver parent cell that 1%B27,10ng/ml bFGF and 10ng/mlBMP4 cultivates 5 days Cell differentiation inducing activities.Again substratum is replaced by DMEM/F12, comprise Insulin-Transferrin-Sodium Selenite mixed solution, 3 μMs of CHIR99021,2 μMs of E-616452, the Ultrapole L of 5 μMs, the S1P of 0.5 μM, the Urogastron of 20ng/mL and people's recombinant albumin of 50 μ g/mL, carry out liver stem cells amplification cultivation.Secondary Culture is carried out with Accutase enzyme when Growth of Cells to 70% converges.
This culture condition can optionally increase human pluripotent stem cells differentiation obtain liver stem cells, and obtain people's liver stem cells under this condition can cultured continuously more than 20 generations.As shown in Figure 4, cultured cells expresses the marker molecule of liver stem cells, as coexpression CD54/DLK1 (Fig. 4 A), AFP/HNF4 (Fig. 4 B), EpCAM/HNF4 (Fig. 4 C), pan-CK/FoxA2 (Fig. 4 D), E-cad/Ki-67 (Fig. 4 E).The positive rate of flowcytometric results showed cell Ki-67 can reach 99%; The positive rate of EpCAM can reach 89% (Fig. 4 F).
Above result shows that the liver stem cells culture system that the present invention sets up can break up the liver parent cell obtained from human pluripotent stem cells the people's liver stem cells that optionally increases, and can maintain the self steady in a long-term of people's liver stem cells in vitro.
Embodiment 3: induction hepatic stem cell differentiation is mature hepatocytes
The liver stem cells of Mice Inoculated or people's vitro culture is in 6 well culture plates (10000 cells/well) of 0.5% matrigel bag quilt.Substratum is DMEM/F12, comprise Insulin-Transferrin-Sodium Selenite mixed solution, 20ng/mL pHGF, 20ng/mL oncostatinM, the transforming growth factor β receptor inhibitor E-616452 of inhibitors of gamma-secretase Compound E and 2 μM of the dexamethasone 1 μM of 100nM.
Through the vitro culture of two weeks, the expression of mature hepatocytes marker molecule was significantly raised; Under this inductive condition, liver stem cells a high proportion ofly can be divided into the mature hepatocytes of expressing and also can secrete ALB; Cell after differentiation has the ability of picked-up acetylated low density lipoprotein, can take in fluorescein-labeled cholic acid and cholic acid is secreted into iuntercellular bile duct simultaneously; Cell after differentiation has the ability (Fig. 5) of urea synthesis.
Above result shows that liver stem cells can be induced to differentiate into the mature hepatocytes with function by hepatic stem cell differentiation system that the present invention sets up efficiently.
Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (8)
1. a long-term in vitro culture system for mouse or people's liver stem cells, is characterized in that, this system is the clear and definite amplification culture medium for cultivating mouse or people's liver stem cells of a kind of Chemical Composition, and described amplification culture medium comprises:
Liquid basal medium,
Insulin-Transferrin-Sodium Selenite the mixed solution of 0.1%-10%,
The glycogen synthase kinase-3 beta inhibitor of 3nM-10 μM,
The transforming growth factor β receptor inhibitor of 0.1 μM-50 μMs,
The Ultrapole L of 0.1 μM-50 μMs,
The S1P of 0.1 μM-50 μMs,
The Urogastron of 0.1-100ng/mL, and
People's recombinant albumin of 0.1-1mg/mL or bovine serum albumin;
Described liquid basal medium, be selected from MEM cell culture medium, DMEM cell culture medium, DMEM/F12 cell culture medium, IMDM cell culture medium, 199 cell culture mediums, F10 cell culture medium, F12 cell culture medium, 1640 cell culture mediums one or more.
2. the long-term in vitro culture system of a kind of mouse according to claim 1 or people's liver stem cells, is characterized in that, described amplification culture medium comprises:
The glycogen synthase kinase-3 beta inhibitor of 0.5 μM-5 μMs;
The transforming growth factor β receptor inhibitor of 1 μM-10 μMs;
The Ultrapole L of 1 μM-10 μMs;
The S1P of 0.1 μM-5 μMs;
The Urogastron of 1-50ng/mL.
3. the long-term in vitro culture system of a kind of mouse according to claim 1 or people's liver stem cells, is characterized in that, described amplification culture medium also comprises: the vitamins C of 0.1-5mM nicotinamide and 0.1-100 μ g/mL.
4. a directed differentiation system for mouse or people's liver stem cells, is characterized in that, this system is the clear and definite division culture medium that liver stem cells can be induced to differentiate into mature hepatocytes of a kind of Chemical Composition, and described division culture medium comprises:
Liquid basal medium,
0.1-100ng/mL pHGF,
0.1-100ng/mL oncostatinM,
The dexamethasone of 10nM-10 μM,
The inhibitors of gamma-secretase Compound E of 10nM-10 μM, and
The transforming growth factor β receptor inhibitor E-616452 of 0.1 μM-50 μMs;
Described liquid basal medium, be selected from MEM cell culture medium, DMEM cell culture medium, DMEM/F12 cell culture medium, IMDM cell culture medium, 199 cell culture mediums, F10 cell culture medium, F12 cell culture medium, 1640 cell culture mediums one or more.
5. the directed differentiation system of a kind of mouse according to claim 4 or people's liver stem cells, is characterized in that, described division culture medium comprises:
5-50ng/mL pHGF;
5-50ng/mL oncostatinM;
The dexamethasone of 50nM-5 μM;
The inhibitors of gamma-secretase Compound E of 100nM-5 μM;
The transforming growth factor β receptor inhibitor E-616452 of 1 μM-20 μMs.
6. the long-term in vitro cultivation of mouse or people's liver stem cells and a method for directed differentiation, it is characterized in that, the method comprises the following steps:
A, utilize as arbitrary in claim 1-3 as described in amplification culture medium, selective amplification liver stem cells from liver parent cell, and vitro culture and going down to posterity is carried out to liver stem cells;
B, utilize division culture medium as described in claim 4 or 5, the liver stem cells that steps A obtains is induced to differentiate into mature hepatocytes.
7. the liver stem cells that long-term in vitro is cultivated and the method for directed differentiation prepares of a mouse as claimed in claim 6 or people's liver stem cells and mature hepatocytes.
8. a liver stem cells as claimed in claim 7 and mature hepatocytes are preparing the application in the treatment cell of hepatic diseases, liver tissue engineering, the external drug screening of hepatic diseases.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410635815.7A CN104388383B (en) | 2014-08-29 | 2014-11-12 | Long-term in-vitro culture and directional differentiation system and method for liver stem cell |
| CN201610997430.4A CN106566799B (en) | 2014-08-29 | 2014-11-12 | A kind of the directed differentiation system and method for liver stem cells |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2014104371335 | 2014-08-29 | ||
| CN201410437133 | 2014-08-29 | ||
| CN201410635815.7A CN104388383B (en) | 2014-08-29 | 2014-11-12 | Long-term in-vitro culture and directional differentiation system and method for liver stem cell |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610997430.4A Division CN106566799B (en) | 2014-08-29 | 2014-11-12 | A kind of the directed differentiation system and method for liver stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104388383A true CN104388383A (en) | 2015-03-04 |
| CN104388383B CN104388383B (en) | 2017-05-24 |
Family
ID=52606344
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610997430.4A Active CN106566799B (en) | 2014-08-29 | 2014-11-12 | A kind of the directed differentiation system and method for liver stem cells |
| CN201410635815.7A Active CN104388383B (en) | 2014-08-29 | 2014-11-12 | Long-term in-vitro culture and directional differentiation system and method for liver stem cell |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610997430.4A Active CN106566799B (en) | 2014-08-29 | 2014-11-12 | A kind of the directed differentiation system and method for liver stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN106566799B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119187A (en) * | 2016-07-01 | 2016-11-16 | 深圳市茵冠生物科技有限公司 | It is hepatocellular culture medium for external evoked adipose-derived Derived from Mesenchymal Stem Cells |
| CN106554936A (en) * | 2015-09-30 | 2017-04-05 | 海门雨霖细胞科技有限责任公司 | Induction new method of the human stem cell to liver cell directed differentiation |
| CN106754659A (en) * | 2016-12-31 | 2017-05-31 | 成都育芽科技有限公司 | A kind of external high activity three-dimensional amplification method of the liver stem cells of maintenance cell phenotype |
| CN108949678A (en) * | 2018-07-26 | 2018-12-07 | 广东卡丝股权投资集团有限公司 | A kind of stem cell media and cultural method |
| CN109251884A (en) * | 2018-10-09 | 2019-01-22 | 刘卫辉 | A kind of method that three-step approach sequential-type induction fetal liver stem cell breaks up to mature hepatocytes |
| CN109837300A (en) * | 2019-03-04 | 2019-06-04 | 山东省千佛山医院 | A method of for improving ALPPS induction hepatic tissue growth rate |
| CN110684717A (en) * | 2019-10-14 | 2020-01-14 | 中国人民解放军第二军医大学 | Culture system for maintaining function of liver cells in vitro for long term and long-term in vitro culture method of mature liver cells |
| WO2020151418A1 (en) * | 2019-01-25 | 2020-07-30 | 中国科学院广州生物医药与健康研究院 | Culture medium for expanding and culturing human liver progenitor cells and application thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107345216B (en) * | 2017-07-28 | 2020-11-06 | 暨南大学 | Adipose-derived stem cell culture medium and application thereof |
| CN110540957B (en) * | 2018-09-21 | 2022-04-29 | 禾美生物科技(浙江)有限公司 | Stem cell induction medium and stem cell induction culture method using same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948413A (en) * | 2011-08-29 | 2013-03-06 | 北京清美联创干细胞科技有限公司 | Hepatic stem cell preserving solution and applications of hepatic stem cell preserving solution |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011096223A1 (en) * | 2010-02-03 | 2011-08-11 | 独立行政法人国立がん研究センター | Induced hepatic stem cell and process for production thereof, and applications of the cell |
| CN101851605B (en) * | 2010-04-29 | 2012-12-05 | 北京弘润天源生物技术有限公司 | Selective medium of liver stem cells, method for selectively separating and amplifying liver stem cells, and medicinal composition for treating diabetes |
| CN102399744A (en) * | 2011-11-24 | 2012-04-04 | 吉林省拓华生物科技有限公司 | Method for culturing liver stem cells |
-
2014
- 2014-11-12 CN CN201610997430.4A patent/CN106566799B/en active Active
- 2014-11-12 CN CN201410635815.7A patent/CN104388383B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948413A (en) * | 2011-08-29 | 2013-03-06 | 北京清美联创干细胞科技有限公司 | Hepatic stem cell preserving solution and applications of hepatic stem cell preserving solution |
Non-Patent Citations (2)
| Title |
|---|
| 王洪山等: "小鼠成体肝脏干细胞体外培养模型的建立", 《中华实验外科杂志》 * |
| 高蕾等: "肝干细胞向成熟肝细胞诱导分化的体内外特点", 《中国组织工程研究与临床康复》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106554936B (en) * | 2015-09-30 | 2020-05-05 | 海门雨霖细胞科技有限责任公司 | New method for inducing human stem cell to directionally differentiate into liver cell |
| CN106554936A (en) * | 2015-09-30 | 2017-04-05 | 海门雨霖细胞科技有限责任公司 | Induction new method of the human stem cell to liver cell directed differentiation |
| CN106119187B (en) * | 2016-07-01 | 2019-07-16 | 深圳市茵冠生物科技有限公司 | It is the culture medium of liver cell for external evoked adipose-derived Derived from Mesenchymal Stem Cells |
| CN106119187A (en) * | 2016-07-01 | 2016-11-16 | 深圳市茵冠生物科技有限公司 | It is hepatocellular culture medium for external evoked adipose-derived Derived from Mesenchymal Stem Cells |
| CN106754659A (en) * | 2016-12-31 | 2017-05-31 | 成都育芽科技有限公司 | A kind of external high activity three-dimensional amplification method of the liver stem cells of maintenance cell phenotype |
| CN106754659B (en) * | 2016-12-31 | 2020-10-30 | 浙江领蔚生物技术有限公司 | Liver stem cell in-vitro high-activity three-dimensional amplification method for maintaining cell phenotype |
| CN108949678A (en) * | 2018-07-26 | 2018-12-07 | 广东卡丝股权投资集团有限公司 | A kind of stem cell media and cultural method |
| CN108949678B (en) * | 2018-07-26 | 2019-08-09 | 广东卡丝股权投资集团有限公司 | A kind of stem cell media and cultural method |
| CN109251884A (en) * | 2018-10-09 | 2019-01-22 | 刘卫辉 | A kind of method that three-step approach sequential-type induction fetal liver stem cell breaks up to mature hepatocytes |
| WO2020151418A1 (en) * | 2019-01-25 | 2020-07-30 | 中国科学院广州生物医药与健康研究院 | Culture medium for expanding and culturing human liver progenitor cells and application thereof |
| CN111607556A (en) * | 2019-01-25 | 2020-09-01 | 中国科学院广州生物医药与健康研究院 | Culture medium for culturing and amplifying human hepatic progenitor cells and application thereof |
| CN111607556B (en) * | 2019-01-25 | 2022-06-07 | 中国科学院广州生物医药与健康研究院 | Culture medium for culturing and amplifying human hepatic progenitor cells and application thereof |
| CN109837300A (en) * | 2019-03-04 | 2019-06-04 | 山东省千佛山医院 | A method of for improving ALPPS induction hepatic tissue growth rate |
| CN110684717A (en) * | 2019-10-14 | 2020-01-14 | 中国人民解放军第二军医大学 | Culture system for maintaining function of liver cells in vitro for long term and long-term in vitro culture method of mature liver cells |
| CN110684717B (en) * | 2019-10-14 | 2021-08-06 | 中国人民解放军第二军医大学 | Culture system for maintaining function of liver cells in vitro for long term and long-term in vitro culture method of mature liver cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104388383B (en) | 2017-05-24 |
| CN106566799B (en) | 2019-08-23 |
| CN106566799A (en) | 2017-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104388383A (en) | Long-term in-vitro culture and directional differentiation system and method for liver stem cell | |
| Ghaedi et al. | Human iPS cell–derived alveolar epithelium repopulates lung extracellular matrix | |
| Huang et al. | The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells | |
| Vosough et al. | Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture | |
| Carpentier et al. | Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen | |
| Emmert et al. | Human stem cell-based three-dimensional microtissues for advanced cardiac cell therapies | |
| Singh et al. | Up-scaling single cell-inoculated suspension culture of human embryonic stem cells | |
| Ghaedi et al. | Alveolar epithelial differentiation of human induced pluripotent stem cells in a rotating bioreactor | |
| Park et al. | Hepatic differentiation of human embryonic stem cells on microcarriers | |
| WO2016045495A1 (en) | Method for in vitro directional differentiation of pluripotent stem cells into cardiac muscle cells | |
| Park et al. | Expansion and hepatic differentiation of rat multipotent adult progenitor cells in microcarrier suspension culture | |
| Kidwai et al. | Differentiation of human embryonic stem cells into clinically amenable keratinocytes in an autogenic environment | |
| EP2723852B1 (en) | Efficient induction of definitive endoderm from pluripotent stem cells | |
| JP7063624B2 (en) | Method for producing liver stem / progenitor cells from mature hepatocytes using small molecule compounds | |
| CA2866590A1 (en) | Defined media for expansion and maintenance of pluripotent stem cells | |
| CN106554936A (en) | Induction new method of the human stem cell to liver cell directed differentiation | |
| Lv et al. | Self‐renewal of hepatoblasts under chemically defined conditions by iterative growth factor and chemical screening | |
| US20110142935A1 (en) | Cardiac differentiation of human pluripotent stem cells under defined conditions using matrix overlay methods | |
| Palakkan et al. | Polarisation and functional characterisation of hepatocytes derived from human embryonic and mesenchymal stem cells | |
| Davila et al. | Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts | |
| Zheng et al. | Regulation of hepatic differentiation of human embryonic stem cells by calcium silicate extracts for liver injury repairing | |
| Raju et al. | The road to regenerative liver therapies: the triumphs, trials and tribulations | |
| JP2016026490A (en) | Culture medium and method for inducing differentiation of pluripotent stem cells to hepatoblasts | |
| CN105121633A (en) | Medium for establishing neuroepithelial stem cells and method and use thereof | |
| Qu et al. | Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |