CN101768571B

CN101768571B - Method for in-vitro inducing and differentiating embryonic stem cells

Info

Publication number: CN101768571B
Application number: CN200910044958XA
Authority: CN
Inventors: 宋后燕; 周鸣鸣
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2009-01-07
Filing date: 2009-01-07
Publication date: 2012-06-06
Anticipated expiration: 2029-01-07
Also published as: CN101768571A

Abstract

The invention belongs to the biotechnology field, relating to a method for inducing and differentiating cells, in particular to a method for in-vitro inducing and differentiating embryonic stem cells, and more specifically relating to a novel method that mouse embryo stem cells are in vitro induced by combination of cytokine and sodium butyrate and efficiently differentiated into liver cells. The experimental results show that the differentiation efficiency of the method can reach 74 percent, higher than the inducement method based on only cytokine or only sodium butyrate in the report of the prior art document.

Description

A kind of method of external evoked, differentiating embryonic stem cells

Technical field

The invention belongs to biological technical field, relate to a kind of method of inducing noble cells.Be specifically related to a kind of embryo stem cell external evoked hepatocellular method that is divided into.More specifically, the present invention relates to that mouse embryo stem cell is external efficiently to be divided into hepatocellular novel method through cytokine and Sodium propanecarboxylate combined induction.

Background technology

Since Thomson in 1998 etc. have reported that (embryonic stemcell ESC) has built since the strain human embryo stem cell, and the research of ESC has caused this area researchist's concern.Because ESC is an isolating multipotential cell system with the of self-replication capacity and totipotency from early stage mammal embryo or archeocyte; The orientable under certain condition cell that is induced to differentiate into almost all kinds; Therefore, its value as cell tissue and organ transplantation new resources soon receives investigator's great attention.Research shows that ESC has broad application prospects in the treatment of congenital hepatopathy, metabolic hepatopathy, but the system of inducing how to set up optimization makes ESC to the differentiation of liver cell direction, then is still the difficult point of present research.

The method of inducing cell differentiation mainly contains 3 kinds: add the cytokine intervention, be total to and cultivate and transgenic.With some tissue-specific silencer in the external source factor activator stem cell; Intracellular signal conduction network is changed; Or strengthen some expression of gene and suppress other expression of gene through transgenic and form the peculiar genetic expression pattern of certain single pedigree, thereby induced dry-cell breaks up to the specific type of tissue cell directional.Yet; Owing at present induce in the atomization gene activation to know little with reticent mechanism and germinal layer, tissue and intercellular information network to ESC; Therefore; Seek new differentiation agent and the induction method of effectively inducing, set up the system of optimizing of inducing make ESC to the liver cell directed differentiation real belong to necessary.

Research has disclosed the ES cell and will experience from entoderm, embryo liver to a plurality of periods such as ripe livers to liver cell differentiation, wherein relates to complicated gene regulating process.Epigenetic modification (epigenetic modification) is reticent and important mechanisms of activated of regulatory gene in the cell, and it comes regulatory gene to transcribe through causing that chromatin and nucleosome configuration change, and does not relate to the change of the sequence of gene own.The epigenetic characteristic is closely related widely in the versatility of stem cell and its born of the same parents, and the new research direction in this field of utilization epigenetic modification strategy induced dry-cell differentiation becoming.

Acetylation of histone and deacetylation are important epigenetic modification modes.Usually the acetylation of histone activated gene is transcribed, and deacetylation then causes gene silencing, and they are controlled by multiple acetylation of histone enzyme and deacetylase respectively.

Sodium propanecarboxylate is a kind of four carbon short chain fatty acids, one butyro-salt, can make the ultra acetylize of histone through the inhibition of histone deacetylase, special silencer in the activating cells, thus cause cytodifferentiation.There are some researches show that Sodium propanecarboxylate can induce fetal rat liver stem cells and human liver cancer cell to break up to ripe liver cell.Up to now, ESC is induced to differentiate into the endoderm cell especially the successful report of liver cell is seldom.

Summary of the invention

The purpose of this invention is to provide a kind of method of external evoked, differentiating embryonic stem cells, be specifically related to a kind of embryo stem cell external evoked hepatocellular method that is divided into.More specifically, the present invention relates to that mouse embryo stem cell is external efficiently to be divided into hepatocellular method through cytokine and Sodium propanecarboxylate combined induction.

The present invention adopts cytokine and Sodium propanecarboxylate combined induction ES cells to break up to liver cell, comprises the steps:

1.ES cells in vitro is cultivated: adopt the Es cell culture medium: do not have the floor height of raising sugar DMEM substratum, behind the ES cell recovery in pretreated 6 orifice plates of gelatin adherent culture, every 1-2d is with 0.25% trysinization and go down to posterity;

2. induce ESC to break up to liver cell: the above-mentioned nutrient solution cultivation that at first adds the removal LIF of people ActivinA in the nutrient solution got restricted endoderm cell in 3 days; In nutrient solution, add people aFGF then, with Sodium propanecarboxylate, combined induction 5 days is to break up to the liver cell direction; Add HGF again; (hepatocyte growth factor HGF) waits inducible factor to continue to induce ripe the cultivation 5 days, at last with OSM; Dex continues to cultivate ripe liver cell 5 days.

Described people ActivinA is 50-150ng/ml, preferred 100ng/ml; Described people aFGF is 20-40ng/ml, preferred 30ng/ml; Described Sodium propanecarboxylate is 2-3mM, preferred 2.5mM; The HGF that adds again is 20-60ng/ml; Described OSM is 10-30ng/ml, and Dex is 0.1-0.4 μ M.

3. liver cell Mark Detection

Comprise the form of observing noble cells; Immunofluorescence detects BSA (albumin; ALB), ALPHA-FP (α-fetoprotein, AFP), (cytokeratin 18, expression CK18) for cytokeratin 18; Detect the expression of specific gene mRNA, the detection of hepatocyte function detection and liver cell differentiation efficiency.

The present invention tests demonstration, and 2.5mmol/L Sodium propanecarboxylate concentration is broken up to liver cell for the most suitable mouse El4 ES cell in combined induction, and concentration crosses that low then differentiation efficiency is low, too highly causes most apoptosis.

The present invention adopts cytokine and Sodium propanecarboxylate combined induction ES cells to break up to liver cell, and its differentiation efficiency is up to 74%, and it is main or simple induction method with Sodium propanecarboxylate with the cytokine that differentiation efficiency is higher than the simple of prior art bibliographical information.

Description of drawings

Fig. 1 is an induction method schema of the present invention

Wherein, ActivinA 100ng/ml; AFGF 30ng/ml; HGF 20ng/ml; Sodiumbutyrate 2.5mM; OSM 10ng/ml; Dex 0.1 μ M.

Fig. 2 is the cellular form after Sodium propanecarboxylate is induced differentiation.

Fig. 3 is that immunofluorescence detects the protein expression result who induces differentiation 18d.

Fig. 4 is the expression of specific gene mRNA.

After 18d was induced in Fig. 5 demonstration, a large amount of Polygons liver cell like cells took on a red color, and showed that cell has the synthetic and storage function of glycogen.

After 18d was induced in Fig. 6 demonstration, ICG male liver cell like cell colony formed, and cellular form is Polygons mostly.

Fig. 7 is the detected result of liver cell differentiation efficiency.

Embodiment

Embodiment 1

1.ES cells in vitro is cultivated: material mouse El4 ES clone is available from the hot university of University of Wisconsin-Madison stem-cell research center.Es cell culture medium: do not have the floor height of raising sugar DMEM substratum, wherein add 10% foetal calf serum, 10U/L LIF (leukemiainhibitory factor.LIF), 0.1mmol/L beta-mercaptoethanol, 1mmol/L Stimulina, non-essential amino acid etc.Behind the ES cell recovery in pretreated 6 orifice plates of gelatin adherent culture, every 1-2d is with 0.25% trysinization and go down to posterity.

2. induce ESC to break up to liver cell: the above-mentioned nutrient solution cultivation that at first adds the removal LIF of people ActivinA 50ng/ml in the nutrient solution obtained restricted endoderm cell in 3 days; In nutrient solution, add people aFGF 20ng/ml and Sodium propanecarboxylate 2mM combined induction 5 days then to break up to the liver cell direction; (hepatocyte growth factor HGF) waits inducible factor to continue to induce ripe the cultivation 5 days to add HGF 20ng/ml again.At last with OSM 10ng/ml; Dex 0.1 μ M continues to cultivate ripe liver cell 5 days.Control group only adds cytokine or only adds Sodium butyrate induces, and spontaneous differentiation group is done negative control group and do not added the above-mentioned factor, only cultivates to let its spontaneous differentiation with the nutrient solution of removing L1F.

3. liver cell Mark Detection

Cellular form is observed: with the form that inverted phase contrast microscope is observed noble cells, Sodium propanecarboxylate is induced differentiation 12d, visible EBs to around the radial cell that scatters partly be the colony growth; Being strand or paving stone appearance changes; It is big that cell volume becomes, polygon or similar round, and the caryoplasm ratio is big; Double-core or three nuclears appear in part, present the form of liver cell like cell.And do not add the control group of cytokine and Sodium propanecarboxylate, and be main then to mix cell, visible volume fibroblast-like cells (like Fig. 2).

Immunofluorescence detects: induce differentiation 18d, the row immunofluorescence dyeing detect BSA (albumin, ALB), ALPHA-FP (α-fetoprotein, AFP), (cytokeratin 18, expression CK18) for cytokeratin 18.The result shows, combined induction differentiation group AFP, ALB, CK18 are all positive, rubescent look or green fluorescence, but the AFP positive cell number is few.

The expression of specific gene mRNA: induce differentiation 6d to rise, RT-PCR detects AAT, the TTR expression in the mRNA level; 12d detects the expression of AFP, ALB, TAT; To differentiation 18d, 6 kinds of special marks of liver cell all have expression.6 kinds of marks of control group normal mouse liver cell all have expression.

Hepatocyte function detects: liver is to carry out the synthetic organ that reaches glyconeogenesis of glycogen, and PAS dyeing can make the interior carbohydrate specific of cell be colored as redness.The result shows, induce 18d after, a large amount of Polygons liver cell like cells take on a red color, and show that cell has the synthetic and storage function of glycogen.The metabolism of ICG is relevant with liver cell basal cell membranes organic anion movement system, and its acceptor is that liver cell is specific, so the picked-up ability of ICG is usually as the specific function sign of liver cell.After inducing 18d, visible ICG male liver cell like cell colony forms, and cellular form is Polygons mostly.

The detected result of liver cell differentiation efficiency shows, breaks up 18d and induces group BSA positive cell percentage ratio by Image-Pro Plus software statistics, obtains inducing group liver cell differentiation rate to be about 74.7% ± 4.9%.Calculate the simple cell factor with method and induce the differentiation group, the liver cell differentiation rate is about 66.3% ± 7.1%.Induce the differentiation group with Sodium propanecarboxylate merely, the liver cell differentiation rate is about 44.5% ± 6.5%.Spontaneous differentiation group liver cell differentiation rate is about 11.1% ± 2.9%.

Embodiment 2

1.ES cells in vitro is cultivated: with embodiment 1.

2. induce ESC to break up to liver cell: the above-mentioned nutrient solution cultivation that adds the removal LIF of people ActivinA 150ng/ml in the nutrient solution obtained restricted endoderm cell in 3 days.In nutrient solution, add people aFGF 40ng/ml and Sodium propanecarboxylate 3mM combined induction 5 days then with to the differentiation of liver cell direction, (hepatocyte growth factor HGF) waits inducible factor to continue to induce ripe the cultivation 5 days to add HGF 60ng/ml again.At last with OSM 30ng/ml; Dex 0.4 μ M continues to cultivate ripe liver cell 5 days.Control group only adds cytokine or only adds Sodium butyrate induces, and spontaneous differentiation group is done negative control group and do not added the above-mentioned factor, only cultivates to let its spontaneous differentiation with the nutrient solution of removing L1F.

3. liver cell Mark Detection method and result are with embodiment 1.

Embodiment 3

1.ES cells in vitro is cultivated: with embodiment 1.

2. induce ESC to break up to liver cell: the above-mentioned nutrient solution cultivation that at first adds the removal LIF of people ActivinA 100ng/ml in the nutrient solution obtained restricted endoderm cell in 3 days.In nutrient solution, add people aFGF 30ng/ml and Sodium propanecarboxylate 2.5mM combined induction 5 days then with to the differentiation of liver cell direction, add inducible factor such as HGF 20ng/ml again and continue to induce ripe the cultivation 5 days.At last with OSM 10ng/ml; Dex 0.1 μ M continues to cultivate ripe liver cell 5 days.Control group only adds cytokine or only adds Sodium butyrate induces, and spontaneous differentiation group is done negative control group and do not added the above-mentioned factor, only cultivates to let its spontaneous differentiation with the nutrient solution of removing L1F.

3. liver cell Mark Detection method and result are with embodiment 1.

Claims

1. the method for external evoked a, differentiating embryonic stem cells is characterized in that adopting cytokine and Sodium propanecarboxylate combined induction ES cells to break up to liver cell, comprises the steps:

1) the ES cells in vitro is cultivated: adopt the Es cell culture medium: do not have the floor height of raising sugar DMEM substratum; Wherein add 10% foetal calf serum, 10U/L LIF (LIF), 0.1mmol/L beta-mercaptoethanol, 1mmol/L Stimulina and non-essential amino acid; Behind the ES cell recovery in pretreated 6 orifice plates of gelatin adherent culture, every 1-2d is with 0.25% trysinization and go down to posterity;

2) induce ESC to break up to liver cell: the above-mentioned nutrient solution cultivation that at first adds the removal LIF of 50-150ng/ml people ActivinA in the nutrient solution got restricted endoderm cell in 3 days; In nutrient solution, add 20-40ng/ml people aFGF then; With 2-3mM Sodium propanecarboxylate combined induction 5 days to break up to the liver cell direction; Add the 20-60ng/mlHGF inducible factor again and continue to induce ripe the cultivation 5 days, at last with 10-30ng/mlOSM; 0.1-0.4 μ M Dex continues to cultivate ripe liver cell 5 days;

3) liver cell Mark Detection

Comprise the form of observing noble cells, immunofluorescence detects the expression of BSA, ALPHA-FP and cytokeratin 18, detects the expression of specific gene mRNA, the detection of hepatocyte function detection and liver cell differentiation efficiency.

2. by the method for described external evoked, the differentiating embryonic stem cells of claim 1, it is characterized in that the described ESC of inducing in the liver cell differentiation step, described people ActivinA is 100ng/ml; People aFGF is 30ng/ml; Sodium propanecarboxylate is 2.5mM.