CN1611599A - Method for inducing stem cell to liver cell directional diferentiation and use of liver cell - Google Patents
Method for inducing stem cell to liver cell directional diferentiation and use of liver cell Download PDFInfo
- Publication number
- CN1611599A CN1611599A CN 200410086475 CN200410086475A CN1611599A CN 1611599 A CN1611599 A CN 1611599A CN 200410086475 CN200410086475 CN 200410086475 CN 200410086475 A CN200410086475 A CN 200410086475A CN 1611599 A CN1611599 A CN 1611599A
- Authority
- CN
- China
- Prior art keywords
- cell
- liver
- stem cell
- differentiation
- hepatocyte
- 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
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 47
- 210000005229 liver cell Anatomy 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 30
- 230000001939 inductive effect Effects 0.000 title claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 102
- 230000004069 differentiation Effects 0.000 claims abstract description 53
- 210000004185 liver Anatomy 0.000 claims abstract description 30
- 238000002360 preparation method Methods 0.000 claims abstract description 15
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 claims description 52
- 230000000763 evoking effect Effects 0.000 claims description 20
- 210000003494 hepatocyte Anatomy 0.000 claims description 20
- 210000004700 fetal blood Anatomy 0.000 claims description 19
- 238000000926 separation method Methods 0.000 claims description 14
- 210000002536 stromal cell Anatomy 0.000 claims description 12
- 210000005087 mononuclear cell Anatomy 0.000 claims description 10
- 239000011324 bead Substances 0.000 claims description 8
- 238000002054 transplantation Methods 0.000 claims description 8
- 210000004024 hepatic stellate cell Anatomy 0.000 claims description 7
- 102000057308 human HGF Human genes 0.000 claims description 6
- 101150052863 THY1 gene Proteins 0.000 claims description 5
- 241001430294 unidentified retrovirus Species 0.000 claims description 5
- 238000007877 drug screening Methods 0.000 claims description 4
- 210000005259 peripheral blood Anatomy 0.000 claims description 4
- 239000011886 peripheral blood Substances 0.000 claims description 4
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 230000001900 immune effect Effects 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 210000004504 adult stem cell Anatomy 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000008929 regeneration Effects 0.000 abstract description 3
- 238000011069 regeneration method Methods 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract 2
- MVORZMQFXBLMHM-QWRGUYRKSA-N Gly-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 MVORZMQFXBLMHM-QWRGUYRKSA-N 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 abstract 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 45
- 239000000047 product Substances 0.000 description 23
- 238000003757 reverse transcription PCR Methods 0.000 description 13
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 12
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 12
- 238000011010 flushing procedure Methods 0.000 description 12
- 102000009027 Albumins Human genes 0.000 description 11
- 108010088751 Albumins Proteins 0.000 description 11
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 9
- 239000004202 carbamide Substances 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 102100033421 Keratin, type I cytoskeletal 18 Human genes 0.000 description 8
- 108010066327 Keratin-18 Proteins 0.000 description 8
- 239000012228 culture supernatant Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 241000283707 Capra Species 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 102100027417 Cytochrome P450 1B1 Human genes 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 101000725164 Homo sapiens Cytochrome P450 1B1 Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 102100036912 Desmin Human genes 0.000 description 4
- 108010044052 Desmin Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 229920001917 Ficoll Polymers 0.000 description 4
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 description 4
- 108010066302 Keratin-19 Proteins 0.000 description 4
- 238000000432 density-gradient centrifugation Methods 0.000 description 4
- 210000005045 desmin Anatomy 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 102100036774 Afamin Human genes 0.000 description 3
- 101710149366 Afamin Proteins 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 3
- 229960003957 dexamethasone Drugs 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000003365 immunocytochemistry Methods 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229920000609 methyl cellulose Polymers 0.000 description 3
- 239000001923 methylcellulose Substances 0.000 description 3
- 229960003966 nicotinamide Drugs 0.000 description 3
- 235000005152 nicotinamide Nutrition 0.000 description 3
- 239000011570 nicotinamide Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102000012466 Cytochrome P450 1B1 Human genes 0.000 description 2
- 108050002014 Cytochrome P450 1B1 Proteins 0.000 description 2
- 108010052832 Cytochromes Proteins 0.000 description 2
- 102000018832 Cytochromes Human genes 0.000 description 2
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- 206010019663 Hepatic failure Diseases 0.000 description 2
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 101000756643 Rattus norvegicus Actin, cytoplasmic 1 Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002440 hepatic effect Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 208000007903 liver failure Diseases 0.000 description 2
- 231100000835 liver failure Toxicity 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 210000004738 parenchymal cell Anatomy 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 239000002435 venom Substances 0.000 description 2
- 231100000611 venom Toxicity 0.000 description 2
- 210000001048 venom Anatomy 0.000 description 2
- 101150033839 4 gene Proteins 0.000 description 1
- 101800000263 Acidic protein Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 208000034423 Delivery Diseases 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 101100260568 Mus musculus Thy1 gene Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 208000020339 Spinal injury Diseases 0.000 description 1
- 231100000643 Substance intoxication Toxicity 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000625 blastula Anatomy 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004992 fission Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000016245 inborn errors of metabolism Diseases 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
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 that to set up a kind of external evoked differentiation of stem cells be 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 stem cell is to the liver cell directed differentiation sophisticated, that function is arranged, liver cell after the directional induction differentiation, for the preparation of hepatocyte transplantation, bioartificial liver and microencapsulated hepatocyte preparation etc. provides seed cell, induce the liver cell of differentiation to can be used for the cell drug screening simultaneously as external pharmacokinetic model.
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 stem cell to the liver cell directed differentiation sophisticated, that function is arranged.
The feeder layer cells of using among the present invention is to infect liver stromal cell system, the feeder layer cells of the stably express human hepatocyte growth factor of foundation with recombinant retrovirus MSCV-HGF.
The liver stromal cell system that uses among the present invention comprises liver Kupffer, hepatic stellate cell.
Stem cell among the present invention comprises people and mammal embryo, marrow, peripheral blood, Cord blood and other tissue-derived mononuclearcell of mobilization, c-Met
+β
2M
-Cell, CD34
+Cell and Thy-1
+Cell.
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.
The present invention has set up the different sources stem cell into hepatocyte and has induced the comparison authentication method that breaks up front and back.
Among the present invention after the external evoked stem cell into hepatocyte directed differentiation liver cell can be used as the preparation that seed cell is used for hepatocyte transplantation, bioartificial liver and microencapsulated hepatocyte preparation etc.
Induce liver cell after the differentiation also to can be used as external pharmacokinetic model among the present invention and be used for the cell drug screening.
Specific embodiments is as follows:
1. separate the adult stem cell of different tissue sources, 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) separation of BMNC: under the aseptic condition, puncture through the bilateral posterior superior iliac spine, gather marrow, 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 BMNC after the density gradient centrifugation is resuspended among the PBS after the washing.
(3) 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 liver stromal cell strain of stably express HGF, the preparation method may further comprise the steps: the liver stromal 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 liver stromal cell strain of stably express HGF, with empty carrier MSCV-neo transfection liver stromal cell, set up the negative control cell strain simultaneously.
3.RT-PCR detect the expression of hHGF mRNA in the liver stromal cell strain of expressing HGF, may further comprise the steps: the total RNA that extracts the liver stromal 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 liver stromal 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. isolating adult stem cell is inoculated on the feeder layer cells, is incubated at and carries out the routine cultivation in the conditioned medium that contains niacinamide, dexamethasone, Regular Insulin etc.
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 the stem cell of inducing differentiation from function, may further comprise the steps: after will inducing stem 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 stem 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 people and mammal embryo, marrow, peripheral blood, Cord blood and other tissue-derived mononuclearcell, CD34
+Cell and Thy-1
+Stem cells such as cell are 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 the preparation that seed cell is applied to hepatocyte transplantation, bioartificial liver and microencapsulated hepatocyte preparation;
3. the liver cell of the external evoked differentiation that obtains according to the technology of the present invention method can be used as the screening that external pharmacokinetic model is used for cell drug.
4. 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:DL2000 Marker; 1: the CFSC/HGF cell of high expression level HGF; 2: low CFSC/HGF cell of expressing HGF; 3: package cell line PT/HGF; The 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:DL2000 Marker; 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.
The specific embodiment
The separation of embodiment 1, human umbilical cord blood mononuclear cell
Through percutaneous umbilical blood sampling, gather health full term natural labor umbilical cord blood under the aseptic and ACD anti-freezing liquid anti-freezing condition, By 4: 1 and 0.5% methylcellulose (U.S., Sigma company product) mixing, sedimented red cell; The sucking-off supernatant partly put from Centrifugal in the core barrel, abandon supernatant after, with the PBS re-suspended cell, add Ficoll (U.S., Sigma company product, relative density 1.077g/ml) collector's human umbilical cord blood mononuclear cell after the 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 rabbit2M antibody (the 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 β2M
-Cell mass; Again with this group cell priority and rabbit Goat anti-rabbit igg (Germany, the Miltenyi of anti-people's C-met antibodies (U.S., Santa Cruz company product) and immunomagnetic beads mark Company's product) hatch after, through the separation and purification of MACS high-gradient magnetic field, finally obtain c-Met+β
2M
-Cell (hCBDCC).
Embodiment 3, rat marrow Thy-1+β
2M
-The separation of cell
Male Fisher344 rats is the taking-up femur after abdomen is cut open, repeatedly flush out bone marrow cell 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-Cell and the rabbit Chinese People's Anti-Japanese Military and Political College mouse Thy-1 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) sorting finally obtains the Thy-1 of rat bone marrow-derived+β2M
-Cell (BDTCs).
The construction step of embodiment 4, human hepatocyte growth factor gene recombinant retroviral vector (MSCV-HGF) is as follows:
(1) also introduce respectively Hpa I and BamH I restriction enzyme site synthetic primer according to the sequence of hHGF-cDNA code area, with The pcDNA3-HGF plasmid is that template is cloned into the HGF plasmid.
(2) will be inserted on the MCS of pMSCVneo carrier with the HGF plasmid of HpaI and BamH I restriction enzyme site, The retroviral vector pMSCV-HGF of genes of interest is carried in acquisition.
(3) confirm that clone's genes of interest sequence is correct, recombinant retrovirus through digestion with restriction enzyme and determined dna sequence Carrier pMSCV-HGF successfully constructs.
Screening and the foundation of the liver stromal cell strain of embodiment 5, efficient, stably express HGF
Take hepatic stellate cells CFSC as example, that the CFSC cellar culture is (complete in the DMEM nutrient solution that contains 10% hyclone Full nutrient solution) in, grow to 40% converge after, to filter the recombinant retrovirus of collecting through 0.45 μ m nylon leaching film PMSCV-HGF venom (polybrene that contains 8 μ g/ml) infects CFSC, after the repeated infection 3 times (every minor tick 24h), Change the fresh culture medium that contains 700 μ g/ml G418 and screen, every 3d changes liquid once, chooses single resistance gram behind the cultivation 14d Grand expansion cultivated, and cell covers with collecting cell culture supernatant behind the 60mm culture dish, detects each clone with the ELISA method and secretes The level of HGF (concrete grammar is referring to the ELISA detection kit specification of hHGF) filters out efficient, stably express HGF The CFSC cell line of (reaching 11.79ng/ml), called after CFSC/HGF. Simultaneously with empty carrier MSCV-neo transfection CFSC, Set up negative control cell strain CFSC/neo.
Embodiment 6, RT-PCR detect the expression of hHGF mRNA among the CFSC/HGF
Extract CFSC, the negative control cell CFSC/neo that infects HGF and produce malicious package cell line with TRIzol reagent Total RNA of PT/HGF carries out with hHGF Auele Specific Primer P1, P2 and rat β-actin Auele Specific Primer P3, P4 respectively RT-PCR, amplified production observe 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
Gene title primer sequence expanding fragment length (bp)
AFP S：5′-TGCAGCCAAAGTGAAGAGGGAAGA-3′ 217
A：5′-CATAGCGAGCAGCCCAAAGAAGAA-3′
Albumin S：5′-TGCTTGAATGTGCTGATGACAGGG-3′ 162
A：5′-AAGGCAAGTCAGCAGGCATCTCATC-3′
CK19 S：5′-ATGGCCGAGCAGAACCGGAA-3′ 328
A：5′-CCATGAGCCGCTGGTACTCC-3′
CYP1B1 S：5′-GAGAACGTACCGGCCACTATCACT-3′ 357
A：5′-GTTAGGCCACTTCAGTGGGTCATGAT-3′
α-tubulin S：5′-CACCCGTCTTCAGGGCTTCTTGGTTT-3′ 528
A：5′-CATTTCACCATCTGGTTGGCTGGCTC-3′
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 (10)
1, a kind of method of external evoked stem cell into hepatocyte directed differentiation, it is characterized in that utilizing stem cell to have the potential of multidirectional differentiation, induce it to the liver cell directed differentiation sophisticated, that function is arranged, for the preparation of hepatocyte transplantation, bioartificial liver and microencapsulated hepatocyte preparation etc. provides seed cell, induce the liver cell of differentiation to can be used for the cell drug screening simultaneously as external pharmacokinetic model.
2, external evoked method according to claim 1 is characterized in that utilizing the feeder layer cells of stably express human hepatocyte growth factor, supports stem cell to the mature liver cells directed differentiation.
3, external evoked method according to claim 2 is characterized in that, described feeder layer cells is to infect liver stromal cell system, the feeder layer cells of the stably express human hepatocyte growth factor of foundation with recombinant retrovirus MSCV-HGF.
4, external evoked method according to claim 3 is characterized in that, described liver stromal cell is to comprise liver Kupffer, hepatic stellate cell.
5, external evoked method according to claim 1 and 2 is characterized in that, described stem cell has multidirectional differentiation potential, comprises peripheral blood, Cord blood and other tissue-derived mononuclearcell, the c-Met of people and mammal embryo, marrow, mobilization
+β
2M
-Cell, CD34
+Cell and Thy-1
+Cell.
6, external evoked method according to claim 5 is characterized in that, described stem cell is the c-Met with the human cord blood source of the separation of indirect immunological magnetic bead sorting method, enrichment and purifying
+β
2M
-Cell.
7, external evoked method according to claim 1 is characterized in that utilizing the hepatic stellate cell strain of stably express human hepatocyte growth factor to make feeder layer cells, the c-Met in backer's Cord blood source
+β
2M
-Cell is to the mature liver cells directed differentiation.
8, external evoked method according to claim 1 and 2 is characterized in that setting up the comparison authentication method that the different sources stem cell into hepatocyte is induced the differentiation front and back.
9, hepatocellular purposes after the external evoked stem cell into hepatocyte directed differentiation is characterized in that inducing the liver cell after the differentiation to can be used as the preparation that seed cell is used for hepatocyte transplantation, bioartificial liver and microencapsulated hepatocyte preparation etc.
10, purposes according to claim 9, its feature also are to induce the liver cell after the differentiation to can be used as external pharmacokinetic model to be used for the cell drug screening.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100864753A CN1303206C (en) | 2003-10-22 | 2004-10-21 | Method for inducing stem cell to liver cell directional diferentiation and use of liver cell |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200310101869.7 | 2003-10-22 | ||
| CN200310101869 | 2003-10-22 | ||
| CNB2004100864753A CN1303206C (en) | 2003-10-22 | 2004-10-21 | Method for inducing stem cell to liver cell directional diferentiation and use of liver cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1611599A true CN1611599A (en) | 2005-05-04 |
| CN1303206C CN1303206C (en) | 2007-03-07 |
Family
ID=34796274
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100864753A Expired - Fee Related CN1303206C (en) | 2003-10-22 | 2004-10-21 | Method for inducing stem cell to liver cell directional diferentiation and use of liver cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1303206C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101148656B (en) * | 2006-09-18 | 2010-10-27 | 中国人民解放军军事医学科学院野战输血研究所 | Construction method for tissue engineering liver unit |
| CN104884072A (en) * | 2012-12-21 | 2015-09-02 | 日东电工株式会社 | Tissue regeneration accelerator |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7432104B2 (en) * | 2001-08-06 | 2008-10-07 | Bresgen Inc. | Methods for the culture of human embryonic stem cells on human feeder cells |
-
2004
- 2004-10-21 CN CNB2004100864753A patent/CN1303206C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101148656B (en) * | 2006-09-18 | 2010-10-27 | 中国人民解放军军事医学科学院野战输血研究所 | Construction method for tissue engineering liver unit |
| CN104884072A (en) * | 2012-12-21 | 2015-09-02 | 日东电工株式会社 | Tissue regeneration accelerator |
| CN104884072B (en) * | 2012-12-21 | 2019-11-08 | 日东电工株式会社 | Regeneration promotor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1303206C (en) | 2007-03-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1195055C (en) | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues | |
| CN101490246B (en) | Isolation and purification of hematopoietic stem cells from post-liposuction lipoaspirates | |
| CN105985985A (en) | Preparation method of allogeneic mesenchymal stem cells edited by CRISPR technology and optimized by IGF (insulin-like growth factor) and application of allogeneic mesenchymal stem cells in treatment of myocardial infarction | |
| CN102876630B (en) | Method for efficiently separating and expanding mesenchymal stem cells in human umbilical cord blood | |
| CN103865876A (en) | Method for primary culture of tumor cells | |
| CN101942413A (en) | Birth defect cell bank and construction method thereof | |
| CN1309180A (en) | Multi-mesoblast pedigree differential potency for stromal cell from fatty tissue, and its use | |
| US20220380733A1 (en) | Method for culturing urine-derived kidney stem cells and use thereof | |
| CN1728946A (en) | Method of obtaining viable human liver cells, including hepatic stem/progenitor cells | |
| WO2010040262A1 (en) | Methods for isolating animal embryonic mesenchymal stem cells and extracting secretion substance thereof | |
| CN104988110A (en) | Method for transforming umbilical cord mesenchymal stem cells into islet cells | |
| CN110484504B (en) | Cell subset for immunotherapy of primary hepatocellular carcinoma and preparation method thereof | |
| CN109679902A (en) | A kind of external evoked amplification method based on cik immunocyte | |
| CN111902533A (en) | Method for producing natural killer cells | |
| CN115386550A (en) | Method for evaluating anticancer effectiveness of immune cells | |
| CN1778905A (en) | Separating culture and use for fatty mesenchymal dry cell | |
| CN105343894A (en) | Recombinant mesenchymal stem cell capable of efficiently secreting prostaglandin E2(PGE2) and application thereof | |
| CN105316286B (en) | A kind of method of preparation and reorganization mescenchymal stem cell and its obtained recombination mescenchymal stem cell | |
| CN116836933B (en) | Liver and gall cancer organoid culture solution, culture reagent combination and culture method | |
| CN104762261A (en) | Tumor infiltrating lymphocytes separation method | |
| CN1303206C (en) | Method for inducing stem cell to liver cell directional diferentiation and use of liver cell | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| CN103239479B (en) | Animal model establishment method of human primary skin squamous epithelial cell cancer stem cell | |
| RU2012107674A (en) | BIOTRANSPLANT FOR RESTORING BONE TISSUE VOLUME AT DEGENERATIVE DISEASES AND TRAUMATIC BONE DAMAGES AND METHOD FOR ITS OBTAINING | |
| CN103305453A (en) | Microcarrier culture system of umbilical cord mesenchymal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20050504 Assignee: SOUTH CHINA CENTER FOR STEM CELL BIOLOGY AND REGENERATIVE MEDICINE OF ACADEMY OF MILITARY MEDICAL SCIENCES Assignor: INSTITUE OF TRANSFUSION MEDICINE, ACADEMY OF MILITARY MEDICAL SCIENCES, PEOPLE'S LIBRATION ARMY OF CHINA Contract record no.: 2015990000123 Denomination of invention: Method for inducing stem cell to liver cell directional diferentiation and use of liver cell Granted publication date: 20070307 License type: Exclusive License Record date: 20150324 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070307 |