CN1303206C

CN1303206C - Method for inducing stem cell to liver cell directional diferentiation and use of liver cell

Info

Publication number: CN1303206C
Application number: CNB2004100864753A
Authority: CN
Inventors: 裴雪涛; 王韫芳; 南雪; 岳�文; 白慈贤; 闫舫
Original assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Current assignee: Institute of Field Blood Transfusion Chinese Academy of Military Medical Sciences
Priority date: 2003-10-22
Filing date: 2004-10-21
Publication date: 2007-03-07
Anticipated expiration: 2024-10-21
Also published as: CN1611599A

Abstract

The present invention relates to biological medicine field, concretely relates to a method for inducing stem cell to liver cell directional differentiation and use of liver cell. The invention combines the genetic engineering with the stem cell engineering, uses the latent energy of the stem cell multiway differentiation, chooses the liver stroma cell for stably expressing human liver cell growth factor, induces the stem cell to liver cell directional differentiation, and induces the fore-and-after stem cell to execute comparative identification. The build of the external induce system not only provides proof for the theory of 'adult stem cell plasticity ', but also can work as seed cell for preparing the stem cell transplant, biological artificial liver and microencapsulation stem cell preparations; the induced and differentiated stem cell can be as external pharmacokinetic model for cell drug screen selecting. The method will has excellent application foreground in regeneration medicine based on genetic engineering, cell engineering and organizing engineering, and will generate great social benefit and economic benefit.

Description

A kind of induced dry-cell is to the method and the hepatocellular purposes of liver cell directed differentiation

Technical field

The present invention relates to biomedical sector, specifically relate to method of a kind of external evoked stem cell into hepatocyte directed differentiation and uses thereof.

Background technology

For the liver failure in whole latter stage that a variety of causes (comprising virus infection, poisonous substance, drug intoxication, tumour, hereditary metabolic disease, liver cancer postoperative etc.) causes, orthotopic liver transplantation is unique effective treatment means at present.Yet, since for extreme shortage, operation and the transplanting of liver itself be correlated with than high mortality, take immunosuppressor throughout one's life and the serious even fatal complication brought makes liver transplantation be subjected to very big restriction in clinical widespread use.Along with stem cell biological is learned deepening continuously of research, be the cell therapy of seed cell with the stem cell, with the stem cell be transport vehicle gene therapy will for whole latter stage the liver failure patient bring new hope.

Stem cell is human body and various histiocytic initial source, has features such as height self and multidirectional differentiation potential and implantable, reconstruction ability.Be the size that these cells can be kept self cell mass by cell fission, can further break up again becomes various histocytes, constitutes the histoorgan of the various complexity of body.Stem cell is divided into myeloid-lymphoid stem cell (as embryonic stem cell, can be divided into all histocytes of body), multipotential stem cell (has multidirectional differentiation potential, can be divided into multiple histocyte, as mescenchymal stem cell etc.) and specially can stem cell (keep the single direction self of a certain particular organization cell, as gut epithelial stem cells etc.).The characteristic that the continuous breakthrough of stem cells technology and stem cell itself are had makes the mankind might be some stem cell of vitro culture, it is divided into our needed various histocytes directional induction, or be used for organizational project for clinical required as seed cell, Stem Cell Engineering with this end in view relates to nearly all vital tissue organ of human body and the human most of difficult medical problem that face, as the treatment of cardiovascular disorder, diabetes, malignant tumour, bone and cartilage defect, senile dementia, Parkinson's disease, burn, Spinal injury and hereditary defect etc.Because the multipotential stem cell in human cord blood, peripheral blood and the marrow, have advantages such as multiplication capacity is strong, the source is abundant, collection is convenient, this type of stem cell transplantation has been brought into play important effect in the treatment of disease in the blood system, malignant tumour, autoimmune disorder etc.

(hepatocyte growth factor HGF) is the actual effect molecule that makes multiple tissue regeneration that extensively distributes in vivo to pHGF.HGF plays a role by combining with surface of cell membrane acceptor c-Met specifically to hepatocyte growth, differentiation, sophisticated support effect.HGF has been widely used in the treatment of clinical disease (as acute liver damage, the hepatic fibrosis of carrying out property etc.) as the cytokine with important biomolecule function, its source mainly is to extract purifying from human plasma and placenta, and this method prepares that HGF is time-consuming, effort, poor activity and cost costliness.And utilize gene engineering expression people HGF to obtain one of effective way with biologic activity HGF in a large number.We have set up the recombinant retroviral vector MSCV-HGF that can stablize, efficiently express HGF, and expectation can make that HGF obtains widely, the application of science.

Liver Kupffer, hepatic stellate cell (hepatic stellate cells, HSCs) be liver stroma cell, can be by producing regeneration of creation such as the soluble cell factor, collagen, collagenase, breeding the microenvironment that plays important support effect to hepatic parenchymal cells.There are some researches show, hepatic parenchymal cells and stroma cell are strengthened its biologic activity according to the meeting of cultivation of certain mixed, prolong the life-span of its growth in vitro.

β ₂M be in the mammalian body wide expression in the karyocyte surface, the albumen of tool conservative property, then do not express this albumen on some tumour cell and embryonic stem cell surfaces of blastula stage with immortality growth characteristics, therefore, β ₂M ^-Cell subsets is considered to have the cell of very strong multiplication capacity; C-Met is the acceptor of HGF, and mediation a series of signal transduction that interacts between HGF and the c-Met plays an important role in mammiferous liver formation and etap, so we think c-Met in marrow ⁺Cell is that a group has the cell to ripe liver cell differentiation potential.In sum, c-Met ⁺β ₂M ^-Cell is that a group is present in the liver ancestral cells in marrow or the Cord blood.

At present, research for liver stem cells has both at home and abroad only confirmed its existence and possible composition, its powerful regeneration and conversion capability have been found, but it really is applied to clinically still remain time, therefore, the adult stem cell that separates different tissue sources, it is divided into liver cell and is used for the preparation of hepatocyte transplantation, bioartificial liver and microencapsulated hepatocyte preparation etc. as seed cell external evoked, induce the liver cell of differentiation to can be used as the screening that external pharmacokinetic model is used for cell drug simultaneously.The foundation of this method will have splendid application prospect in the regenerative medicine based on genetically engineered, cell engineering and even organizational project etc., and will have a tremendous social and economic benefits.

Summary of the invention

Technical problem to be solved by this invention is to set up a kind of external evoked Cord blood c-Met ⁺β ₂M ^-Cytodifferentiation is hepatocellular method.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

Utilize stem cell to have the potential of multidirectional differentiation, external evoked Cord blood c-Met ⁺β ₂M ^-Cell is to the liver cell directed differentiation sophisticated, that function is arranged, the liver cell after the directional induction differentiation, and the preparation that can be hepatocyte transplantation or external biological artificial liver provides seed cell.

The external evoked method of using among the present invention is a feeder layer cells of utilizing the stably express human hepatocyte growth factor, supports Cord blood c-Met ⁺β ₂M ^-Cell is to the liver cell directed differentiation sophisticated, that function is arranged.

The feeder layer cells of using among the present invention is the recombinant retrovirus MSCV-HGF infection hepatic stellate cell with the expressing human pHGF, the feeder layer cells of the stably express human hepatocyte growth factor of foundation.

C-Met among the present invention ⁺β ₂M ^-Cell is the stem cell with the human cord blood source of the separation of indirect immunological magnetic bead sorting method, enrichment and purifying.

The hepatic stellate cell strain that the present invention utilizes is stable, efficiently express human hepatocyte growth factor is as feeder layer cells, but the c-Met in backer's Cord blood source ⁺β ₂M ^-Cell is to the liver cell directed differentiation sophisticated, that function is arranged.

Among the present invention after the external evoked stem cell into hepatocyte directed differentiation liver cell can be used as preparation or the hepatocyte transplantation that seed cell is used for the external biological artificial liver.

Specific embodiments is as follows:

1. separate Cord blood c-Met ⁺β ₂M ^-Cell, the preparation method is:

(1) separation of Cord blood mononuclearcell: get blood through umbilical vein puncture under the aseptic and ACD anti-freezing liquid anti-freezing condition, gather healthy full-term normal delivery umbilical cord blood, successively through the 0.5% methylcellulose gum (U.S., Sigma company product) sedimentation and the Ficoll (U.S., Sigma company product, relative density 1.077g/ml) collector's Cord blood mononuclearcell after the density gradient centrifugation is resuspended among the PBS after the washing.

(2) Cord blood c-Met ⁺β ₂M ^-The separation of cell: get the human cord blood mononuclearcell that is resuspended among the PBS, successively with the anti-people β of rabbit ₂After M antibody (U.S., Santa Cruz company product) and the anti-rabbit igg of immunomagnetic beads labelled goat (Germany, Miltenyi company product) are hatched,, obtain β through MACS high-gradient magnetic field (Germany, Miltenyi company product) separation and purification ₂M ^-Cell mass; After again this group cell successively being hatched with the anti-people c-Met of rabbit antibody (U.S., Santa Cruz company product) and the anti-rabbit igg of immunomagnetic beads labelled goat (Germany, Miltenyi company product),, finally obtain c-Met through the separation and purification of MACS high-gradient magnetic field ⁺β ₂M ^-Cell.

2. set up efficient, the hepatic stellate cell strain of stably express HGF, the preparation method may further comprise the steps: the hepatic stellate cell routine is incubated in the DMEM nutrient solution (complete culture solution) that contains 10% foetal calf serum, grow to 40% converge after, to filter recombinant retrovirus pMSCV-HGF venom (polybrene that the contains 8 μ g/ml) cells infected of collecting through 0.45 μ m nylon leaching film, after the repeated infection 3 times (each 24h at interval), changing the fresh substratum that contains 700 μ g/ml G418 screens, every 3d changes liquid once, choose single resistance clone enlarged culturing after cultivating 14d, cell covers with collecting cell culture supernatant behind the 60mm culture dish, detect the level (concrete grammar is referring to the ELISA detection kit specification sheets of hHGF) of each clone's secretion HGF with the ELISA method, filter out efficient, the hepatic stellate cell strain of stably express HGF, with empty carrier MSCV-neo transfection hepatic stellate cell, set up the negative control cell strain simultaneously.

3.RT-PCR detect the expression of hHGF mRNA in the hepatic stellate cell strain of expressing HGF, may further comprise the steps: the total RNA that extracts the hepatic stellate cell strain, negative control cell and the product poison package cell line PT/HGF that infect HGF with TRIzol reagent, carry out RT-PCR with hHGF Auele Specific Primer and rat β-actin Auele Specific Primer respectively, amplified production is observed the situation of transcribing of hHGF mRNA through 1% agarose gel electrophoresis.

4. external enlarged culturing is expressed the hepatic stellate cell of HGF, treats that cell grows to acceptance when converging more than 90% ⁶⁰Feeder layer cells is set up in Co gamma-rays 18Gy irradiation.

5. with isolating human cord blood c-Met ⁺β ₂M ^-Cell inoculation is incubated at and carries out the routine cultivation in the conditioned medium that contains niacinamide, dexamethasone, Regular Insulin etc. on feeder layer cells.

6. under the inverted microscope external evoked stem cell is compared evaluation from morphology.

7. identify liver cell specific gene and proteic expression with RT-PCR, immunocytochemistry from genetic transcription and protein translation level respectively, may further comprise the steps: extract the cell total rna of inducing the differentiation front and back with TRIzol reagent, use different primers (seeing Table 1) to carry out RT-PCR and 1% agarose gel electrophoresis respectively, observe the situation of transcribing of mRNA; With induce after the differentiation stem cell and as the cell of feeder layer with the PBS flushing after, with acetone in 4 ℃ fixedly behind the 20min, anti-and fluorescein-labeled two anti-hatching with corresponding respectively, PBS flushing back is observation of cell dyeing situation under fluorescent microscope.

8. Fox Green absorbs, the excretion function by detecting respectively, indexs such as albumin, urea secretion function compare evaluation to inducing the cell after the differentiation from function, may further comprise the steps: after will inducing cell after the differentiation with the PBS flushing, add 1mg/mlICG in 37 ℃ hatch 15min after, microscopically is observed; After PBS flushing 2 times, gain the conventional 4h of cultivation of perfect medium after, observe once more; Induce the cell after the differentiation to use the DMEM substratum of serum-free instead, add the NH of 0.15mol/L through the PBS flushing ₄Cl, collecting cell culture supernatant behind the conventional cultivation 8h detects urea and albuminous secretory volume in the culture supernatant on automatic clinical chemistry analyzer.

The invention has the beneficial effects as follows;

1. according to technological method provided by the invention, can be with the c-Met in human cord blood source ⁺β ₂M ^-Cell is divided into the liver cell sophisticated, that function is arranged at directional induction in vitro;

2. the liver cell of the external evoked differentiation that obtains according to the technology of the present invention method can be used as preparation or hepatocyte transplantation that seed cell is applied to the external biological artificial liver;

3. the foundation of method of inducing differentiation will have splendid application prospect among the present invention in the regenerative medicine based on genetically engineered, cell engineering and even organizational project etc., and will have a tremendous social and economic benefits.

Description of drawings

Fig. 1 RT-PCR detects the expression of hHGF mRNA in CFSC/HGF and the CFSC/neo cell.

M:DL2000Marker; 1: the CFSC/HGF cell of high expression level HGF; 2: low CFSC/HGF cell of expressing HGF; 3; Package cell line PT/HGF 4:CFSC/neo cell.

①HGF 396bp；②β-actin 516bp。

The CFSC/HGF cell of Fig. 2 vitro culture and the hCBDCC that induces front and back.

2a CFSC/HGF cell monoclonal; The CFSC/HGF cell of 2b enlarged culturing;

2c is inoculated in the hCBDCC on the CFSC/HGF cell feeder layer; 2d hCBDCC induced the 8th day.

Fig. 3 RT-PCR method detects the expression that hCBDCC induces front and back liver cell specific gene mRNA.

M:DL2000Marker; 1:HepG2 clone; 2: human foetus liver cell;

Before 3:hCBDCC induces; After 4:hCBDCC induces.

①CYP1B1；②AFP；③Alb；④CK18；⑤α-tubulin

Fig. 4 immunofluorescence chemical detection result

4a-d is that hCBDCC induces the 8th day alpha-fetoprotein (AFP) and albumin (Alb) immunofluorescence detected result;

4e-h is CFSC/HGF cell GFAP and Desmin immunofluorescence detected result.

Fig. 5 Fox Green picked-up experiment.

The output of albumin, urea in Fig. 6 cells and supernatant

1: normal liver cell; 2:hCBDCC induced the 8th day; 3:hCBDCC induced the 4th day;

4:hCBDCC induced the 0th day; The 5:CFSC/HGF cell.

Embodiment

The separation of embodiment 1, Cord blood mononuclearcell

Get blood through umbilical vein puncture under the aseptic and ACD anti-freezing liquid anti-freezing condition, gather healthy full-term normal delivery umbilical cord blood, by 4: 1 and 0.5% methylcellulose gum (U.S., Sigma company product) mixing, sedimented red cell; The sucking-off supernatant is partly put in the centrifuge tube centrifugal, abandon supernatant after, with the PBS re-suspended cell, add collector's Cord blood mononuclearcell after Ficoll (U.S., Sigma company product, the relative density 1.077g/ml) density gradient centrifugation, be resuspended among the PBS after the washing.

The c-Met in embodiment 2, human cord blood source ⁺β ₂M ^-The separation of cell (hCBDCC)

Get the human cord blood mononuclearcell that is resuspended among the PBS, successively with the anti-people β of rabbit ₂After M antibody (U.S., Santa Cruz company product) and the anti-rabbit igg of immunomagnetic beads labelled goat (Germany, Miltenyi company product) are hatched,, obtain β through MACS high-gradient magnetic field (Germany, Miltenyi company product) separation and purification ₂M ^-Cell mass; After again this group cell successively being hatched with the anti-people c-Met of rabbit antibody (U.S., Santa Cruz company product) and the anti-rabbit igg of immunomagnetic beads labelled goat (Germany, Miltenyi company product),, finally obtain c-Met through the separation and purification of MACS high-gradient magnetic field ⁺β ₂M ^-Cell (hCBDCC).

Embodiment 3, rat marrow Thy-1 ⁺β ₂M ^-The separation of cell

Male F344 rat is taken out femur after abdomen is cut open, flush out medullary cell repeatedly with complete culture solution, after 200 eye mesh screens filter, be resuspended among the PBS, (ρ=1.083g/ml) density gradient centrifugation obtains the rat marrow mononuclearcell through Ficoll, successively with the rabbit Chinese People's Anti-Japanese Military and Political College mouse β 2M antibody (U.S., Santa Cruz company product) and the goat anti-rabbit igg (Germany of immunomagnetic beads mark, Miltenyi company product) hatch after, through MACS high-gradient magnetic field (Germany, Miltenyi company product) separation and purification obtains β 2M ^-Cell; Again with the β 2M that collects ^-The cell and the rabbit Chinese People's Anti-Japanese Military and Political College mouse Thy-1 antibody (U.S., Santa Cruz company product) and after the goat anti-rabbit igg of immunomagnetic beads mark (Germany, Miltenyi company product) hatches, through MACS high-gradient magnetic field (Germany, Miltenyi company product) sorting finally obtains the Thy-1 that rat marrow is originated ⁺β ₂M ^-Cell (BDTCs).

The structure of embodiment 4, human hepatocyte growth factor gene recombinant retroviral vector (MSCV-HGF)

Step is as follows:

(1) also introducing Hpa I and BamH I restriction enzyme site synthetic primer respectively according to the sequence of hHGF-cDNA coding region, is that template is cloned into the HGF plasmid with the pcDNA3-HGF plasmid.

(2) the HGF plasmid that will have Hpa I and a BamH I restriction enzyme site is inserted on the multiple clone site of pMSCVneo carrier, obtains to carry the retroviral vector pMSCV-HGF of goal gene.

(3) confirm that through digestion with restriction enzyme and determined dna sequence clone's target gene sequences is correct, recombinant retroviral vector pMSCV-HGF successfully constructs.

The screening and the foundation of the liver stromal cell strain of embodiment 5, efficient, stably express HGF

With hepatic stellate cell is that CFSC is an example, the CFSC routine is incubated in the DMEM nutrient solution (complete culture solution) that contains 10% foetal calf serum, grow to 40% converge after, infect CFSC to filter the recombinant retrovirus pMSCV-HGF venom of collecting (polybrene that contains 8 μ g/ml) through 0.45 μ m nylon leaching film, after the repeated infection 3 times (each 24h at interval), changing the fresh substratum that contains 700 μ g/ml G418 screens, every 3d changes liquid once, choose single resistance clone enlarged culturing after cultivating 14d, cell covers with collecting cell culture supernatant behind the 60mm culture dish, detect the level (concrete grammar is referring to the ELISA detection kit specification sheets of hHGF) of each clone's secretion HGF with the ELISA method, filter out efficient, the CFSC cell strain of stably express HGF (reaching 11.79ng/ml), called after CFSC/HGF.With empty carrier MSCV-neo transfection CFSC, set up negative control cell strain CFSC/neo simultaneously.

Embodiment 6, RT-PCR detect the expression of hHGF mRNA among the CFSC/HGF

Extract CFSC, negative control cell CFSC/neo that infects HGF and the total RNA that produces malicious package cell line PT/HGF with TRIzol reagent, carry out RT-PCR with hHGF Auele Specific Primer P1, P2 and rat β-actin Auele Specific Primer P3, P4 respectively, amplified production is observed the situation of transcribing of hHGF mRNA through 1% agarose gel electrophoresis.

P1：5′-GTTGTCCCTGTATGCCTCTG-3′；P2：5′-GAGCCAGGGCAGTAATCTC-3′；

P3：5′-GTTGTCCCTGTATGCCTCTG-3′；P4：5′-GAGCCAGGGCAGTAATCTC-3′。

Found that, express the CFSC of HGF and the amplified band that 396bp appears in the PT/HGF cell, and the cell CFSC/neo of infection empty carrier does not see positive band that confirmation hHGF gene is transcribed (see figure 1) in the purpose cell.

Embodiment 7, hCBDCC carried out on feeder layer routine is cultivated and morphological observation

The CFSC/HGF cell is pressed 5 * 10 ⁵/ ml is inoculated in the 60mm culture plate conventional the cultivation, behind the 20h with ⁶⁰Co gamma-rays 18Gy irradiation.The hCBDCC of separation and purification is pressed 3 * 10 ⁵/ ml inoculation is incubated in the inducing culture that contains niacinamide, dexamethasone, Regular Insulin etc. thereon.Observe down in inverted phase contrast microscope behind the conventional 4～8d of cultivation, find that part hCBDCC volume obviously increases, occur big and single even a plurality of nucleus circle, kytoplasm is abundant, and division mutually more to be seen.

Embodiment 8, the hCBDCC of external evoked differentiation is identified from phenotype and function

4.1.RT-PCR method detect alpha-fetoprotein (AFP), albumin (albumin, Alb), the expression of cytokeratin 18 (CK18), Cytochrome P450 1B1 (CYP1B1)

Extract the cell total rna of inducing the differentiation front and back with TRIzol reagent, use following primer (seeing Table 1) to carry out RT-PCR and 1% agarose gel electrophoresis respectively, observe the situation of transcribing of mRNA.Found that, induce the preceding cell of differentiation not express CK18 and CYP1B1, weak expression AFP and Alb, differentiation back cell is all expressed above-mentioned 4 gene (see figure 3)s.

Table 1 RT-PCR primer title, sequence and expanding fragment length

The gene title	Primer sequence	Expanding fragment length (bp)
The gene title	Primer sequence	Expanding fragment length (bp)	AFP Albumin CK19 CYP1B1 α-tubulin	S：5′-TGCAGCCAAAGTGAAGAGGGAAGA-3′ A：5′-CATAGCGAGCAGCCCAAAGAAGAA-3′ S：5′-TGCTTGAATGTGCTGATGACAGGG-3′ A：5′-AAGGCAAGTCAGCAGGCATCTCATC-3′ S：5′-ATGGCCGAGCAGAACCGGAA-3′ A：5′-CCATGAGCCGCTGGTACTCC-3′ S：5′-GAGAACGTACCGGCCACTATCACT-3′ A：5′-GTTAGGCCACTTCAGTGGGTCATGAT-3′ S：5′-CACCCGTCTTCAGGGCTTCTTGGTTT-3′ A：5′-CATTTCACCATCTGGTTGGCTGGCTC-3′	217 162 328 357 528

4.2. immunocytochemistry detects the expression of alpha-fetoprotein (AFP), albumin (Alb), neuroglia fibres acidic protein (GFAP) and desmin (Desmin)

With induce differentiation 8d hCBDCC and as the CFSC/HGF cell of feeder layer with the PBS flushing after, with acetone in 4 ℃ fixedly behind the 20min, anti-and fluorescein-labeled two anti-hatching with corresponding one respectively, PBS flushing back is observation of cell dyeing situation under fluorescent microscope.Found that c-Met ⁺β ₂M ^-Express AFP and Alb after the cell induction differentiation and feeder layer cells expression GFAP and Desmin (see figure 4).

4.3. Fox Green (ICG) absorbs, the detection of excretion function

With the hCBDCC that induces differentiation 8d with after the PBS flushing, add 1mg/ml ICG in 37 ℃ hatch 15min after, microscopically is observed the hCBDCC that volume increases and is dyed deep green, and unconverted hCBDCC of form and feeder layer cells color no change.After PBS flushing 2 times, gain the conventional 4h of cultivation of perfect medium after, the color fade (see figure 5) of cytochrome.

4.4. urea and albuminous output in the cells and supernatant

Induce the hCBDCC of differentiation 8d to wash the DMEM substratum of using serum-free instead, add the NH of 0.15mol/L through PBS ₄Cl, collecting cell culture supernatant behind the conventional cultivation 8h detects urea and albuminous secretory volume in the culture supernatant on automatic clinical chemistry analyzer.Found that c-Met ⁺β ₂M ^-Cell is along with the prolongation of induction time, and albumin and urea production increase (see figure 6) gradually.

Embodiment 9, rat BDTCs carried out on feeder layer routine is cultivated and morphological observation

The CFSC/HGF cell is pressed 5 * 10 ⁵/ ml is inoculated in the 60mm culture plate conventional the cultivation, behind the 20h with ⁶⁰Co gamma-rays 18Gy irradiation.BDTCs is pressed 5 * 10 ⁴/ cm ²Inoculation is incubated in the conditioned medium that contains niacinamide, dexamethasone, Regular Insulin etc. thereon.Observe down in inverted phase contrast microscope behind the conventional 4～8d of cultivation, find that the part cell volume increases from cultivating the 4th day, obviously increase to the 7th day volume, occur big and single, two even a plurality of nucleus circle, endochylema is abundant, and the part cell is in the division phase.

Embodiment 10, the BDTCs of external evoked differentiation is identified from phenotype and function

4.1.RT-PCR method detect hepatocyte neclear factor (HNF) 3 β, alpha-fetoprotein (AFP), albumin (albumin, Alb), the expression of cytokeratin 18 (CK18), cytokeratin 19 (CK19), Cytochrome P450 1B1 (CYP1B1)

Extract the cell total rna of inducing the differentiation front and back with TRIzol reagent, carry out RT-PCR and 2% agarose gel electrophoresis, observe the situation of transcribing of mRNA.Found that, induce the preceding cell of differentiation not express HNF3 β, CK18, CK19 and CYP1B1, weak expression AFP and Alb, differentiation back cell is all expressed above-mentioned 6 genes.

4.2. immunocytochemistry detects the expression of albumin (Alb) and Keratin sulfate (CK18)

With induce differentiation 7d BDTCs and as the CFSC/HGF cell of feeder layer with the PBS flushing after, with acetone after 4 ℃ fixedly 30min and 0.1%Triton X-100/PBS are hatched 20min, successively with antialbumin antiserum(antisera) and FITC mark two anti-and with two anti-hatching of anti-CK18 antibody and TRITC mark, PBS flushing back is observation of cell dyeing situation under fluorescent microscope.Found that Alb and CK18 are expressed in cell induction differentiation back.

4.3. Fox Green (ICG) absorbs, the detection of excretion function

In inducing system, add 1mg/ml ICG in 37 ℃ hatch 15min after, microscopically is observed the BDTCs that volume increases and is dyed deep green, and unconverted BDTCs of form and feeder layer cells color no change.After PBS flushing 2 times, gain the conventional 4h of cultivation of perfect medium after, the color fade of cytochrome.

4.4. urea and albuminous output in the cells and supernatant

Induce the BDTCs of differentiation 7d to wash the DMEM substratum of using serum-free instead, add the NH of 0.15mol/L through PBS ₄Cl, collecting cell culture supernatant behind the conventional cultivation 8h detects urea and albuminous secretory volume in the culture supernatant on automatic clinical chemistry analyzer.Found that under the support of CFSC/HGF BDTCs has and similar amino metabolism generation urea of normal liver cell and the albuminous function of secretion after inducing differentiation.

Claims

1. the c-Met+ β 2M-cell in a human cord blood source is to the method for liver cell directional induction differentiation, it is characterized in that utilizing stem cell to have the characteristics of multidirectional differentiation potential, the c-Met+ β 2M-cell of inducing the human cord blood source under the support of feeder layer cells is to the liver cell differentiation sophisticated, that function is arranged.

2. directional induction differentiation method according to claim 1, it is characterized in that utilizing stable, as to efficiently express human hepatocyte growth factor hepatic stellate cell strain as feeder layer cells, the c-Met+ β 2M-cell in backer's Cord blood source is to the mature liver cells directed differentiation.

3. directional induction differentiation method according to claim 1 and 2, it is characterized in that, described feeder layer cells is the recombinant retrovirus MSCV-HGF infection hepatic stellate cell with the expressing human pHGF, the feeder layer cells of the stably express human hepatocyte growth factor of foundation.

4. directional induction differentiation method according to claim 1 is characterized in that the c-Met+ β 2M-cell that utilizes the separation of indirect immunological magnetic bead sorting method, enrichment and purifying human cord blood to originate.

5. the hepatocellular application that obtains of the described directional induction differentiation method of claim 1 is characterized in that the liver cell of inducing differentiation that obtains provides seed cell for hepatocyte transplantation or bioartificial liver's preparation.