CN104388383B - 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
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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, and in particular to the long-term in vitro culture of a kind of liver stem cells and fixed
To differentiated system and method, including a kind of clear and definite amplification culture medium of chemical analysis, for the external of mouse or people's liver stem cells
Culture；And a kind of clear and definite differential medium of chemical analysis, for mouse or people's liver stem cells to be induced to differentiate into ripe liver
Cell.
Background technology
The annual patient dead because of hepatopathy of China is available for liver transplantation to be only capable of meeting 5% hepatopathy every year more than 300,000
The demand of patient.It is available for the deficient clinical practice for seriously limiting the treatment means of liver transplantation.It is existing preclinical and face
Bed is studied and shown, can be replanted in liver injury after mature hepatocytes or liver stem cell transplantation and be rebuild liver, but either
Mature hepatocytes or liver stem cells equally exist the deficient problem in source.
Being by human pluripotent stem cells (embryonic stem cell and induced pluripotent stem cells) Differentiation Induction in vitro can stabilization in vitro
The liver stem cells of culture not only can provide the cell derived of stabilization for the cell transplantation of hepatopath, and avoid using tire liver group
Involved ethnics Problem is knitted, advantage is apparent.But the directional induction of current multipotential stem cell is all mostly by many
The atomization of the timing of step, finally obtains functional, cell (the Wobus AM ＆ Boheler KR of terminal differentiation
Embryonic Stem Cells:Prospects for Developmental Biology and Cell
Therapy.Physiol.Rev.2005；85(2):635-678.) at present, differentiated system cannot be still stably maintained at specific
The middle stage of development, such as the tissue stem cell stage.Meanwhile, at present also without being effectively induced to differentiate into human pluripotent stem cells
The method that the liver stem cells of amplification can be stablized.
Therefore, this area still lacks effective means amplifying liver stem cells, especially cannot be in the culture of specific chemical components
Under the conditions of realize liver stem cells such as long-term in vitro culture and directed differentiation.
The content of the invention
Long-term in vitro culture and directed differentiation system it is an object of the invention to provide a kind of liver stem cells, the system bag
A kind of clear and definite amplification culture medium of chemical analysis is included, for mouse or the in vitro culture of people's liver stem cells；And a kind of chemistry into
Part clearly differential medium, for mouse or people's liver stem cells to be induced to differentiate into mature hepatocytes.
A kind of method another object of the present invention is to provide the long-term in vitro culture and directed differentiation of liver stem cells, should
Human pluripotent stem cells can be induced to differentiate into liver stem cells by method, and liver stem cells can be maintained to expand steadily in the long term and keep
Directed differentiation is the ability of mature hepatocytes.
The first aspect of the present invention, is to provide the long-term in vitro cultivating system of a kind of mouse or people's liver stem cells, the system
It is a kind of amplification culture medium that chemical analysis is explicitly used for cultivating mouse or people's liver stem cells, described amplification culture medium bag
Include：
Liquid basal medium,
0.1%-10% (volumn concentration) Insulin-Transferrin-sodium selenite mixed liquor,
3nM-10 μM of glycogen synthase kinase-3 beta inhibitor,
0.1 μM -50 μM of transforming growth factor β receptor inhibitor,
0.1 μM -50 μM of lysophosphatidic acid,
0.1 μM -50 μM of S1P,
The EGF of 0.1-100ng/mL,
With people's recombinant albumin or bovine serum albumin(BSA) of 0.1-1mg/mL.
Described liquid basal medium, selected from MEM cell culture mediums, DMEM cell culture mediums, the training of DMEM/F12 cells
Support base, IMDM cell culture mediums, 199 cell culture mediums, F10 cell culture mediums, F12 cell culture mediums, 1640 cell culture mediums
In one or more.
Preferably, described amplification culture medium contains 0.5 μM -5 μM of glycogen synthase kinase-3 beta inhibitor；
Preferably, described amplification culture medium contains 1 μM -10 μM of transforming growth factor β receptor inhibitor；
Preferably, described amplification culture medium contains 1 μM -10 μM of lysophosphatidic acid；
Preferably, described amplification culture medium contains 0.1 μM -5 μM of S1P；
Preferably, described amplification culture medium contains the EGF of 1-50ng/mL.
Preferably, described amplification culture medium also includes：The vitamin C of 0.1-5mM niacinamides and 0.1-100 μ g/mL.
Described glycogen synthase kinase-3 beta inhibitor can be CHIR99021 or other small molecule glycogen synthase kinase-3s β
Inhibitor；
Described transforming growth factor β receptor inhibitor can be E-616452 or other transforming growth factor β receptors
Inhibitor.
The 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 the system is one
Liver stem cells can be clearly induced to differentiate into the differential medium of mature hepatocytes, described differentiation culture to plant chemical analysis
Base includes：
Liquid basal medium,
0.1-100ng/mL HGFs,
0.1-100ng/mL oncostatinMs,
10nM-10 μM of dexamethasone,
10nM-10 μM of inhibitors of gamma-secretase Compound E, and
0.1 μM -50 μM of transforming growth factor β receptor inhibitor E-616452.
Described liquid basal medium, selected from MEM cell culture mediums, DMEM cell culture mediums, the training of DMEM/F12 cells
Support base, IMDM cell culture mediums, 199 cell culture mediums, F10 cell culture mediums, F12 cell culture mediums, 1640 cell culture mediums
In one or more.
Preferably, described differential medium contains 5-50ng/mL HGFs；
Preferably, described differential medium contains 5-50ng/mL oncostatinMs；
Preferably, described differential medium contains 50nM-5 μM of dexamethasone；
Preferably, described differential medium contains 100nM-5 μM of inhibitors of gamma-secretase Compound E；
Preferably, described differential medium contains 1 μM -20 μM of transforming growth factor β receptor inhibitor E-616452.
The third aspect of the present invention, is to provide long-term in vitro culture and the directed differentiation of a kind of mouse or people's liver stem cells
Method, the method is comprised the following steps：
A, using foregoing amplification culture medium, the selective amplification liver stem cells from liver mother's (Hepatoblasts) cell,
And in vitro culture and passage are carried out to liver stem cells；
B, using foregoing differential medium, the liver stem cells that step A is obtained are induced to differentiate into mature hepatocytes.
In step A, described liver stem cells liver mother cell is from embryo liver in mice tissue or by human pluripotent stem cells
What differentiation was obtained.
Liver mother cell stem cell is obtained from embryo liver in mice tissue, general knowledge is known in the art, reference can be made to document：
Weiss MC,Strick-Marchand H.Isolation and characterization of mouse hepatic
stem cells in vitro.Semin Liver Dis 2003；23:313-324..
Liver mother cell stem cell is obtained by human pluripotent stem cells differentiation, general knowledge is known in the art, reference can be made to document：
Takayama K,Nagamoto Y,Mimura N,Tashiro K,Sakurai F,Tachibana M,Hayakawa T,et
al.Long-Term Self-Renewal of Human ES/iPS-Derived Hepatoblast-like Cells on
Human Laminin 111-Coated Dishes.Stem Cell Reports 2013；1:322-335.
The method of the present invention, can obtain liver mother cell from embryo liver in mice tissue or by human pluripotent stem cells differentiation
Middle selective amplification liver stem cells, by liver stem cells long-term in vitro culture, and Induction of committed differentiation is mature hepatocytes.
The fourth aspect of the present invention, be to provide according to the long-term in vitro culture of foregoing a kind of mouse or people's liver stem cells and
Liver stem cells and mature hepatocytes that the method for directed differentiation is prepared.
The fifth aspect of the present invention, the liver stem cells and mature hepatocytes being to provide according to are preparing liver diseases
Application in treatment cell, liver tissue engineering, the external drug screening of liver diseases.
Beneficial effects of the present invention：Can be from small using the clear and definite liver stem cells amplification culture medium of chemical analysis of the invention
Mouse fetal liver tissue breaks up selective amplification liver stem cells, liver stem cells in acquisition liver mother cell by human pluripotent stem cells
Can with this understanding cultivate more than 20 generations, and maintain the liver stem cells molecular phenotype of stabilization；The method of the present invention is without using magnetic
Pearl or complicated, high cost the cell purification means of airflow classification, the liver stem cells purity cultivated can reach 99%.
The mouse of culture or people's liver stem cells can be lured using chemical analysis of the invention clear and definite hepatic stem cell differentiation culture medium
Lead that be divided into can Albumin Secretion, the mature hepatocytes of the function such as metabolizing urea.
Brief description of the drawings
Fig. 1 is using the clear and definite amplification cultivation based selective culture mice embryonic liver stem cells of chemical analysis, cell culture
To the 5th generation, i.e., cannot detect the expression (A) of endothelial cell and interstitial cell gene；And EpCAM positive liver stem cells
Purity can reach 98.5% (B)；Cell culture can still keep normal caryogram (C), and stable gene expression profile to 20 generations
(D)。
Fig. 2 is that the cell of culture has the phenotype of liver stem cells, the marker molecule of expression liver stem cells, is such as co-expressed
Albumin (ALB) and keratin 19 (K19), liver precursor label DLK1, EpCAM and CD133, liver endogenous retroviral because
Sub- HNF4 α and FoxA2, while epithelial cell molecular marked compound E-Cadherin and β-Catenin (A) that is co-expressed；Fluidic cell
Art analysis result display cell expresses CD133 (99% positive rate), Ki-67 (88% positive rate), DLK1 (94% positive rate),
And K19 (99%positive) (B)；Immunofluorescence dyeing result shows that the single liver stem cells of culture have two-way differentiation
Potential, can be divided into the positive liver cells of ALB and the positive bile duct epithelial cells (C) of K19.Fig. 3 is the liver stem cells of culture
The bile duct spline structure formed in three-dimensional Matrigel, A and B is that the bile duct observed under phase contrast microscope supports structure, and B is A
The enlarged drawing of rectangle frame in figure；The bile duct that C is arrived for transmission electron microscope observation；Immunocytochemical stain shows the courage to be formed
Pipe spline structure expresses the marker molecule (D, E, F) of bile duct.
The liver stem cells that the differentiation of Fig. 4 cultures human pluripotent stem cells is obtained, the mark point of the cell expression liver stem cells of culture
Son, such 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 display cell Ki-67 can reach 99%；The positive rate of EpCAM can reach
89% (F).
Fig. 5 is that hepatic stem cell differentiation is mature hepatocytes, and by the in vitro culture of two weeks under the conditions of induction, liver does
Cell can (A, B and C, B be the amplification of rectangle frame in A figures with a high proportion of mature hepatocytes for being divided into expression and can secreting ALB
Figure)；Cell after differentiation has the ability (D) of intake acetylated low density lipoprotein, while can take in fluorescein-labeled
Cholic acid is simultaneously secreted into iuntercellular bile duct (E) by cholic acid；Cell after differentiation has the ability (F) of urea synthesis.
Specific embodiment
With reference to drawings and Examples of the invention to it is of the invention implementation elaborate, following examples be with
Implemented under premised on technical solution of the present invention, given detailed implementation method and specific operating process, but the present invention
Protection domain be not limited to following embodiments.
Embodiment 1：Amplification mice embryonic liver stem cells
The mice embryonic of pregnancy 12.5 days is taken, fetal liver is taken out, is digested with Accutase enzymes (Invitrogen companies)
Afterwards, single cell suspension is inoculated in 0.5%Matrigel matrigels (BD Bioscience companies) or 20 μ g/ml
Laminin (Invitrogen companies) coated 6 well culture plate, culture medium is DMEM/F12 (Invitrogen companies), comprising
Insulin-Transferrin-sodium selenite mixed liquor (Invitrogen companies), 3 μM of CHIR99021 (Tocris companies), 2 μM
E-616452 (Sigma-Aldrich companies), 5 μM of lysophosphatidic acid (Sigma-Aldrich companies), 0.5 μM of 1- phosphoric acid
Sphingol (Sigma-Aldrich companies), the EGF (R＆D Systems companies) of 20ng/mL and the people of 50 μ g/mL
Recombinant albumin (R＆D Systems companies).Cell growth to 70% converge when carry out Secondary Culture with Accutase enzymes.
The condition of culture can selectivity amplification embryonic hepatic stem cells, as shown in figure 1, no longer can after the generation of cell culture five
Detect blood vessel endothelium and fibroblastic in the presence of (Figure 1A), and the purity of liver stem cells can reach more than 95% (figure
1B).Liver stem cells with continuous passage more than 20 generations, and can keep cell caryogram (Fig. 1 C) and gene expression profile with this understanding
Stabilization (Fig. 1 D).
The CMC model cell express liver stem cells marker molecule (Fig. 2), such as coexpression Albumin (ALB) and
Keratin 19 (K19), liver precursor label DLK1, EpCAM and CD133, liver endogenous transcription factor HNF4 α and FoxA2,
Be co-expressed epithelial cell molecular marked compound E-Cadherin and β-Catenin (Fig. 2A) simultaneously.Flowcytometric results show
Show that 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 that the single liver stem cells of culture have two-way differentiation potential, can be divided into ALB
Positive liver cell and the positive bile duct epithelial cells (Fig. 2 C) of K19.The liver stem cells of culture can be with three-dimensional Matrigel
Form bile duct spline structure (Fig. 3 A, B).The inner cavity surface of electron microscope observation display bile duct cell has substantial amounts of microvillus (figure
3C)；And the bile duct spline structure for being formed expresses the marker molecule of bile duct, such as K19, Integrin α 6, Integrin β 4 and Aqu1 etc.
(Fig. 3 D, E, F).
Result above show the present invention foundation liver stem cells cultivating system can from embryo liver in mice selectivity
Amplifying liver stem cells, it is possible to maintain the self-renewing steady in a long-term of liver stem cells in vitro.
Embodiment 2：The liver stem cells that amplification human pluripotent stem cells differentiation is obtained
Human pluripotent stem cells H1 and Hues9 cell (Wicell companies) are incubated at the coated 6 hole culture of 0.5% matrigel
Plate, culture medium is DMEM/F12, comprising 1%N2 (Invitrogen companies), 1%B27 (Invitrogen companies) and 20ng/
Ml bFGF (Invitrogen companies).DMEM/F12 is replaced medium to during cell growth to 50% fusion, B27 is (without pancreas islet
Element), it is endoderm cell that 100ng/ml Activin A (R＆D Systems companies) cultivate four days Cell differentiation inducing activities；Then
DMEM/F12 is replaced medium to, comprising 1%N2,1%B27,5 μM of SB431542 (Sigma-Aldrich companies) and 10ng/
Ml BMP4 (Invitrogen companies) 2 days inducing cells of culture enter a part and turn to veutro anterior intestine；Then replace medium to
DMEM/F12, it is female liver to cultivate 5 days Cell differentiation inducing activities comprising 1%N2,1%B27,10ng/ml bFGF and 10ng/mlBMP4
Cell.Culture medium is replaced by DMEM/F12 again, comprising Insulin-Transferrin-sodium selenite mixed liquor, 3 μM
CHIR99021,2 μM of E-616452,5 μM of lysophosphatidic acid, 0.5 μM of S1P, the epidermal growth of 20ng/mL
People's recombinant albumin of the factor and 50 μ g/mL, carries out liver stem cells amplification cultivation.Cell growth to 70% converge when use
Accutase enzymes carry out Secondary Culture.
The condition of culture can selectivity amplification human pluripotent stem cells differentiation obtain liver stem cells, and obtain people liver
Stem cell can be continuously cultivated more than 20 generations under this condition.As shown in figure 4, the mark point of the cell expression liver stem cells of culture
Son, such as coexpression CD54/DLK1 (Fig. 4 A), AFP/HNF4 (Fig. 4 B), EpCAM/HNF4 (Fig. 4 C), pan-CK/FoxA2 (figure
4D), E-cad/Ki-67 (Fig. 4 E).The positive rate of flowcytometric results display cell Ki-67 can reach 99%；
The positive rate of EpCAM can reach 89% (Fig. 4 F).
Result above shows what the liver stem cells cultivating system of present invention foundation can be obtained from human pluripotent stem cells differentiation
In liver mother cell selectivity amplification people's liver stem cells, it is possible in vitro maintain people's liver stem cells it is steady in a long-term self more
Newly.
Embodiment 3：Induction hepatic stem cell differentiation is mature hepatocytes
(10000 is thin in coated 6 well culture plate of 0.5% matrigel for the liver stem cells of Mice Inoculated or people's in vitro culture
Born of the same parents/hole).Culture medium is DMEM/F12, comprising Insulin-Transferrin-sodium selenite mixed liquor, 20ng/mL hepatic cell growths
The factor, 20ng/mL oncostatinMs, the inhibitors of gamma-secretase Compound E of 1 μM of the dexamethasone of 100nM and 2 μM of conversion
Growth factor beta receptor inhibitor E-616452.
By the in vitro culture of two weeks, the expression of mature hepatocytes marker molecule was significantly raised；The liver under this inductive condition
Stem cell can be with a high proportion of mature hepatocytes for being divided into expression and can secreting ALB；Cell after differentiation has intake acetyl
Change the ability of low-density lipoprotein, while fluorescein-labeled cholic acid can be taken in and cholic acid is secreted into iuntercellular bile duct；Point
Cell after change has the ability (Fig. 5) of urea synthesis.
Result above shows that liver stem cells can be induced efficiently the hepatic stem cell differentiation system of the present invention foundation differentiation
It is tool functional mature hepatocytes.
Below the preferred embodiment to the invention is illustrated, but the invention be not limited to it is described
Embodiment, those of ordinary skill in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (4)
1. the long-term in vitro cultivating system of a kind of mouse or people's liver stem cells, it is characterised in that the system is a kind of chemical analysis
It is explicitly used for cultivating the amplification culture medium of mouse or people's liver stem cells, described amplification culture medium includes：
Liquid basal medium,
The Insulin-Transferrin of 0.1%-10%-sodium selenite mixed liquor,
3nM-10 μM of glycogen synthase kinase-3 beta inhibitor,
0.1 μM -50 μM of transforming growth factor β receptor inhibitor,
0.1 μM -50 μM of lysophosphatidic acid,
0.1 μM -50 μM of S1P,
The EGF of 0.1-100ng/mL, and
People's recombinant albumin or bovine serum albumin(BSA) of 0.1-1mg/mL；
Described liquid basal medium, selected from MEM cell culture mediums, DMEM cell culture mediums, DMEM/F12 cell culture mediums,
In IMDM cell culture mediums, 199 cell culture mediums, F10 cell culture mediums, F12 cell culture mediums, 1640 cell culture mediums one
Plant or two or more.
2. the long-term in vitro cultivating system of a kind of mouse according to claim 1 or people's liver stem cells, it is characterised in that institute
The amplification culture medium stated includes：
0.5 μM -5 μM of glycogen synthase kinase-3 beta inhibitor；
1 μM -10 μM of transforming growth factor β receptor inhibitor；
1 μM -10 μM of lysophosphatidic acid；
0.1 μM -5 μM of S1P；
The EGF of 1-50ng/mL.
3. the long-term in vitro cultivating system of a kind of mouse according to claim 1 or people's liver stem cells, it is characterised in that institute
The amplification culture medium stated also includes：The vitamin C of 0.1-5mM niacinamides and 0.1-100 μ g/mL.
4. the long-term in vitro cultural method of a kind of mouse or people's liver stem cells, it is characterised in that the method is comprised the following steps：
Using the amplification culture medium as described in claim 1-3 is any, the selective amplification liver stem cells from liver mother cell, and it is right
Liver stem cells carry out in vitro culture and passage.
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| 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 |
| 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 |
| 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 |
| CN107345216B (en) * | 2017-07-28 | 2020-11-06 | 暨南大学 | Adipose-derived stem cell culture medium and application thereof |
| 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 |
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| 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 |
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