Generation Of Human Induced Pluripotent Stem Cells Of Chronic Hepatic Failure Patients

Somatic cell can be induced into Induced Pluripotent Stem Cell(iPS) by specific transcription factor and it will be constantly update, proliferation and multiplex differentiation. In August,2006, mice fibroblasts were induced into iPS for the first time using foure transciption factors—Oct4, Sox2, C-Myc and Klf4by the Japanese scholars Yamanaka. In2007, human somatic cells were induced into iPS by Yamanaka and Thomson research group successively. After that, study and attention about iPS was growth explosive all of the world, and also some breakthrough wsa made, such as building a specific iPS cell line of people disease, building iPS cell line of no virus by transgene, using small molecules to get high efficiency iPS cells, in addition rats and monkeys iPS cells had been established. Because iPS cells are not restricted to cell sources, immune rejection, ethics, religious and legal, all these will make iPS cells for clinical early, iPS cells research and applications will be expected to become greatest achievements of medical biology in the twentyfirst centurey.Liver transplantation can significantly reduce mortality in patients with liver failure, but due to the serious shortage of donor liver, the fact that only a third of patients can undergo liver transplant, and most of the patients are death during the process of waiting for donor liver. Based on this premise, domestic and foreign scholars design the different types of artificial liver system to create conditions about liver cell regeneration and liver function recovery for liver failure patients, thus reduce mortality in patients. Liver support about liver failure patients is almost entirely depend on the role of the liver cell biology function. Cell source of artificial liver is more, but At present, there is no cell for completely needing for clinical.Because iPS cell is similar to embryonic stem cell(ES cells) which is constantly renew, constantly proliferation and multiplex differentiation potential, also not be restricted to cells sources, immune rejection, ethics, religious and legal restrictions, we want to make fibroblasts, using tissue explant technique to get fibroblasts from chronic hepatic failure patients(CHF),induce into iPS cell by retroviruses carrier with Oct4, Sox2, Klf4and c-Myc genes in order to attain the following goals:(1) Master iPS cells’programming technolog in order to explore cells to provide technical support for artificial liver system to treat chronic hepatic failure patients;(2) Establish human iPS cell line to research its biological characteristics and its mechanism during induction proces;(3)Find new cell sources for artificial liver system. This experiment includes the following three research content:1.CF-1mouse embryos fibroblasts feeder layer preparation2.Retroviruses preparation and identification3.Human iPS cells line establish and identificationObjectiveInduced fibroblasts,which using using tissue explant technique to get from chronic hepatic failure patients into iPS by retroviruses carrier with Oct4, Sox2, Klf4and c-Myc genes in order to attain the following goals.Methods1.CF-1mouse embryos fibroblasts feeder layer preparationPut pregnant13.5day’s CF-1rat to death by cervical, take out fetal rat sterily, remove its head、limbs and internal organsaseptic, keep only its trunk.Cut trunk into fragment and add10mL of trypsin which is isopyknic mixing by0.25%and0.05%,then put it in37℃,5%CO2,95%humidity incubator15min, every3.0-4.0minutes gently blow1.0min. Add isopyknic MEF medium to terminate digestion, and then into10cm dishe (about each dishe with one embryo). Treated the third generation of it with different concentrations (5,10,15,20mg/L) of mitomycin C for different time periods (1,1.5,2,2.5,3hours) to prepare feeder layers, and observe its proliferation and cryopreserved with1×105per freeze-stored tube.2.Retroviruses preparation and identificationRetroviruses production Get amount of about Oct4, Sox2,Klf4,c-Myc EGFP and virus packaging plasmids which are provided by Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences with QIANGEN plasmid extraction kit, and then according to the nucleic acid calcium chloride.being in the form of DNA sediment, can make DNA attach to cell surface, which is beneficial to engulfed by cell. Produce retroviruses with293-T cell according to Calcium chloride mediated transfection program, collect virus after packaging48h and save at-80℃after its concentration measurement respectively.Retroviruses concentration determination Infection150000of293T cells with5μl virus about48h,fixed wth formaldehyde,dyeing with DAPI, count EGFP positive cells and total cell number with fluorescence microscope and bringing it to the formula:virus drops degrees (IU/ml)=EGFP positive cell count/total cell count÷5×1.5×105×103.3.reprogramming chronic hepatic failure patients skin fibroblasts(cHF) to iPS cell by Retroviruses(1) After filtrate with0.45u m filter, transfection chronic hepatic failure patients skin fibroblasts with a certain amount of the virus(2) After24h about virus infection,digest fibroblasts and plant into6orifice plate according to1×104/hole,the6orifice plate has been with feeder cell.On the tenth day, digest fibroblasts and plant into10cm dish according to10×104/10cm per dish,and then with iPS cells conditions medium.(3) About20days, choose AP positive cloning to continue to culture.4. Identification of human iPS cell line(1) Morphology under microsocopy and take pictures(2) Alkaline phosphatase(AP) dyeing(3) Immunocytochemistry was used to detect the expression of hEScell marker genes(4) Total RNA was isolated,and RT-PCR was used to detect the expression of hES cell marker genes(5) The karyotype analysis of cHF derived hiPS cells(6) Suspension culture to observe the embryo formation and the differentiation in vitro(7) Teratoma fomation by transplanting CHFF derived hiPS cells into SCID miceResults:1. CF-1mouse embryos fibroblasts feeder layer preparationThe optimal fetal age for preparing CF-1mouse embryo fibroblast feeder layer was13.5days. Treatment with10mg mitomycin C for2.5hours showed optimal effects on inhibiting the proliferation of CF-1mouse embryo fibroblasts and fibroblast feeder layer could maintain5-7days. Induced pluripotent stem cells and embryonic stem cells can develop into typical "bird nest"-shaped colony of stem cells after treatment with10mg/L mitomycin C for2.5hours.2. Retroviruses preparation and identificationOct4,Sox2,c-Myc,KIf4,EGFP and retroviruses packaging plasmids were induced into293-t cell to product virus by calcium reprogramme method.After48hours, green fluorescent can be observed in the EGFP control group,which explain that transfection was success and the virus could transfection eukaryocyte.And the concentration of virus was above1×06IU/ml.3. Reprogramming chronic hepatic failure patients skin fibroblasts(cHF) to iPS cell by RetrovirusesWe used each2ml of virus to transfect cHF and grew them on feed layer after48hours.48-72hours after infection.green fluorescenec was observed under fluorescence microscopy,the morphogoly of cHFF changed7-8days after infection,about10days, we used iPS cells conditioned medium and about20days,we picked AP positive colonies.4. Identification of human iPS cell line(1) The morphogoly of cHFF changed7days after infection,we used iPS cells conditioned medium and about20days.we picked AP positive colonies.(2) Two lines of iPS cells had morphological characteristics of hES cells and alkaline phosphatase activity proved positive;(3) Two lines of iPS cells expressed hES cell-specific marker genes(4) Two lines of iPS cells expressed hES cell-specific markers:SSAE-4, SSAE-1,TRA-1-60,TRA-1-81,OCT4;(5) Karyotype of cHF derived hiPS cells was normal;(6) cHF derived hiPS cells foemed embryoid bodies(Ebs) in vitro,and Ebs could be spontaneous differentiation by adherent culture;(7) Two lines of iPS cells could form Teratomas,and expressed the specific marker genes of three germ layers.Conclusion1. The optimal fetal age for preparing CF-1mouse embryo fibroblast feeder layer was13.5days. Treatment with10mg mitomycin C for2.5hours showed optimal effects on inhibiting the proliferation of CF-1mouse embryo fibroblasts and fibroblast feeder layer could maintain5-7days. Induced pluripotent stem cells and embryonic stem cells can develop into typical "bird nesf"-shaped colony of stem cells after treatment with10mg/L mitomycin C for2.5hours.2. The concentration of virus is above1×106IU/ml by calcium packaging system.The best infection plural of virus is2ml/6orificeplate-hole and it can induce eukaryotic cells.The cHF can be induced into iPS with Oct4, Sox2, Klf4, and c-Myc four genes. And two lines of hiPS have some of the biological characteristics to ES cells,which indicates that we have had cHF derived hiPS cell line.