Generation Of Human Induced Pluripotent Stem Cells From Umbilical Cord Matrix And Human Skin Fibroblasts

Background&ObjectiveCardiovascular diseases are the number one killer for human health in today’s world, accounting for35%of cause of death of human disease. Myocardial infarction and heart failure is the leading cause of death of cardiovascular disease. Because of coronary artery occlusion and interruption of cardiac blood supply, Myocardial infarction lead to myocardial cell injury and necrosis. Heart failure is cardiac myocyte contraction and (or) diastolic dysfunction, decreased cardiac output. Myocardial infarction is the main causes of heart failure. Coronary artery occlusion lead to myocardial infarction. Myocardial necrosis is in the center of infracted zone, the ischemic injury and apoptosis of myocardial cell is around the infracted zone. Because the number of myocardial cells decrease and the cells lack of proliferation and differentiation capacity, myocardial cell infarction area will be filled by the fibroblasts, the area form scar tissue. The ventricular wall thinning, aneurysm formation, ventricular remodeling, systolic and diastolic dysfunction, cardiac electrical activity disorder and other factors will eventually leads to patients death. Currently, the myocardial necrosis or fibrosis are not recovered to be normal myocardial cells function through thrombolytic therapy, percutaneous coronary intervention and coronary artery bypass graft surgery, cardiac failure resynchronization therapy. The heart transplantation is the most effective method for terminal cardiac failure, but there are many problems during treatment process, such as a shortage of donor organs, the high cost of medicine, high standards of surgery, autoimmune rejection issues, therefore it is restricted during clinical application process. Stem cells is a kind of undifferentiated potential cells, it has self-renewal, infinite proliferation and multiplex differentiation potential. Stem cells can repair the damage and necrosis of myocardial cell. It will brings new hope for patients with cardiovascular disease,such as myocardial infarction, heart failure, cardiomyopathy, complete atrioventricular block and sick sinus syndrome.In1981, Evnas and Martin respectively established mouse embryonic stem cell lines. Thomson and Shamblott used fertilization blastocysts and miscarriage embryo to establish embryo human embryonic stem cell in1998. Further studies found that human embryonic stem cells can be induced into nerve cells, smooth muscle cells, myocardial cells, hematopoietic cells, more than200kinds of cells, etc. In1999, Bjornson differentiated neural stem cells of mouse of central nervous system into blood cells. It means that the adult stem cells also has horizontal differentiation potential.According to the different developmental stages of stem cells, it can be divided into embryonic stem cells and adult stem cells. The adult stem cells include hematopoietic stem cells, bone marrow mesenchymal stem cells, muscle stem cells, mesenchymal stem cells, adipose stem cells etc. According to the differentiation potential of stem cells, it can be divided into versatile stem cell, pluripotent stem cells and single stem cells. Embryonic and adult stem cells are two candidate stem cells of transplantation in the treatment of cardiovascular diseases. Embryonic stem cells are pluripotential stem cells, which can be differentiated into multiple types of myocardial cells. Because embryonic stem cells has ethics controversy, so far it is only applied to animal experiments, not in clinical trials. Adult stem cells is pluripotent stem cells, it can be differentiated into myocardial cells under certain conditions in vitro. The adult stem cells transplant of patients not only overcome its own immune rejection, but also avoids the ethical debate. It is the seed cells to treat cardiovascular disease, and having many problems in clinical treatment process.Cell reprogramming means that somatic cells can be reversed to an undifferentiated state. The reversal cells become to be pluripotential stem cells, which can be induced to many kinds of cells. In recent years, induced pluripotent stem cells is focus in the field of stem cell research. In August2006, Japan’s Kyoto university,Yamanaka research team first induced mouse fibroblasts into pluripotential stem cells, which can be called "iPS cells". iPS cells means that somatic cells is reprogrammed to be embryonic stem like-cells. iPS cells form, surface antigen, gene expression, proliferation and differentiation potentials are similar to embryonic stem cells. It not only avoids the political and ethical controversy, but also immune rejection reaction, therefore iPS cells provides new ideas and methods for stem cell treatment of cardiovascular diseases. Establishing iPS cells include the following main steps:Choosing somatic and reprogramming gene, transfecting exogenous gense into somatic cells, the cultivation of viral infections of somatic cell, pick and identification of iPS cells, etc.2009Scientists in China got a mice from iPS cells with tetraploid blastocyst injection, proved the iPS cells pluripotency. The research results indicate that iPS cells can replace embryonic stem cells for the treatment of many diseases, but also proved that iPS cells can differentiate into all types of heart cells for the stem cells treatment of cardiovascular diseases. There are the following characteristics as seed cells for the stem cell treatment of cardiovascular diseases:①Source of adequate, easily obtained and accepted patients.②After transplantation, it is a good biological safety for patients, who has no own immune rejection.③No ethical and social controversy. First, obtained the umbilical cord matrix cells through a enzyme digestion method, human skin fibroblasts through a skin tissue adherent method. Secondly, the umbilical cord matrix cells and human skin fibroblasts will be induced into iPS cells with Oct-4, Sox-2, Klf-4and c-Myc genes, their gene transfer vector is a retroviral-mediated system. Finally, biological identification of iPS cells include cell gene expression, karyotype analysis, human ES cell-specific protein detection, differentiation of teratomas in vivo, differentiation of embryoid bodies in vitro. Our objectives are to master technology of somatic cell reprogramming and establish iPS cells from the umbilical cord matrix cells and human skin fibroblasts. Our next objective is to differentiate iPS cells into cardiomyocytes and vascular endothelial cells.Methods1Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Human Skin Fibroblasts.1.1Obtained and cultured UMC and HSF cells:Taking umbilical cord and skin from newborn baby or patient, we will isolate and cultivate UMC cells from umbilical cord with the enzyme digestion method.HSF cells will be isolated and cultivated from patients skin by tissue adherent method.1.2Preparation of CF-1mouse embryonic fibroblast feeder:We isolated embryonic tissues from13.5days pregnant CF-1mouse. After cuting embryonic tissues into pieces and then adding0.15%trypsin, they were digested for5minutes in37℃、5%CO2incubator. P3generation of mouse embryonic fibroblasts were processed in3hours in370C、5%CO2incubator, after added mitomycin C working solution of10ug/ml concentration into them. Finally, the MEF feeder cells were frozen for future applications.1.3Retrovirus packaged and producted:Recovery of cryopreserved293T cells, the cells were inoculated into five dishes when their density is100%. It is about80%-85%of growth density before plasmid transfected cells. pMX-hOCT-4, pMX-hSOX-2, pMX-hKLF-4, pMX-hc-MYC, pMX-GFP and pCL packaging plasmids were transfected into293T cells by calcium phosphate method.293T cell culture medium was replaced in12-16hours.In36-48hours UMC and HSF cells were infected with retrovirus packaged and producted.1.4UMC and HSF cells were infected with retrovirus collected:Recovery of frozen UMC and HSF cells. Accoding to4×104/each hole density, the UMC and HSF cells were divided into a six-well plate in the plate. We collected retroviral supernatant, and then they was filtered through0.45um membrane filter and added polybrene (8ug/ml) to mixing.After that, the UMC and HSF cells are infected by retroviral supernatant.1.5Infected UMC and HSF cells were cultured:Cell culture medium will be replaced to DFBS medium in2day after cells infected. In6days UMC and HSF cells were seeded and cultured in MEF feeder.In18day DFBS medium will be replaced to KSR medium.1.6ES-like iPS cells clones were picked:We recover MEF feeder cells to a12well plates before picking iPS cells. The next day, the cells medium was replaced to the KSR medium. The cells room was UV disinfection for30minutes before picking iPS cell clones. The iPS cell clones will be selected under the microscope if their morphology look like ES cells. We will cut and suck iPS cell clones, and then transfer these clones into a12well plates paved with MEF feeder cells.1.7The iPS cells will be cultured in the MEF feeder or no-feeder culture system: After picking iPS cell clones, they will be purified and cultured in the MEF feeder. And then the iPS cells will be inoculated and cultured in no-feeder culture system. We will extract DNA and RNA of iPS cells to detect expression levels of the gene.2Identification of iPS cells2.1Detecting gene expression:For iPS cells, exogenous gene (Sox-2, Klf-4, Oct-4, c-Myc) and endogenous genes (Oct4, Sox2. Nanog, Rex1) expression are detected with RT-PCR method.2.2Identification of gel electrophoresis of exogenous genes:Exogenous gene (Sox-2, Klf-4, Oct-4, c-Myc) is inserted into the iPS cells and identified by the gel electrophoresis.2.3Karyotype analysis:We will analyze iPS cell karyotype and judge whether iPS cells have normal karyotype.2.4Alkaline phosphatase staining and the expression of ES cell-specific protein: For iPS cells, alkaline phosphatase is detected by alkaline phosphatase staining, ES cell membrane protein expression (SSEA3, SSEA4), matrix protein (TRA-1-60, TRA-1-81), nucleoprotein (Nanog) are detected by immunofluorescence staining.2.5Expression of ES cell-specific protein:For iPS cells, ES cell membrane protein expression (SSEA3, SSEA4), matrix protein (TRA-1-60, TRA-1-81), nucleoprotein (Nanog) are detected by immunofluorescence staining.2.6Nanog, Oct4methylation sequencing:Nanog, Oct4methylation status will be detected by application of DNA methylation sequencing. These genes are methylation or demethylation.If Nanog. Oct4are demethylation, it means that they are reacted and express.2.7iPS cells will be differentiated into teratoma in vivo:The iPS cells will be injected into SCID mice back and inner thighs. If there is teratoma formation two months later, we examine whether iPS cells have ability to differentiate the three germ layers or not through teratoma biopsy. 2.8iPS cells will be differentiated into embryoid body in vivo:iPS cells will be differentiated into embryoid body during cultivation in vivo. We will extract RNA of embryoid body cells.Gene expression level were detected by RT-PCR, which include endoderm gene (AFP, GATA4, SOX17), the mesoderm genes (TBX1), ectodermal genes (PAX6, SOX1).Results1Establishing iPS cells from UMC and HSF cells.1.1UMC and HSF cells isolated and cultivated in vitro:With the enzyme digestion and tissue adherent method, we successfully obtained UMC and HSF cells from the umbilical cord and skin tissuse.1.2Preparation of CF-1mouse embryonic fibroblast feeder:P3generation of MEF cells were processed with mitomycin C for3hours. We observed MEF under a microscopy.The MEF morphology unchanged. They had no a lot of death. The number of cells not increased.These results indicated that the cell proliferation was inhibited while while maintaining viability.1.3Retrovirus packaged and producted:The exogenous genes (Sox-2, Klf4, Oct-4, c-Myc) were tranfected into293T cells with a calcium phosphate method. The293T cells packaged and producted retrovirus with exogenous genes.1.4UMC and HSF cells were infected with retrovirus collected:We collected retroviruses supernatant fluid to infect UMC and HSF two times.The retroviruses successfully infected UMC and HSF cells.1.5Infected UMC and HSF cells were cultivated:After the UMC and HSF cells were infected with retroviruses for4-5days,their morphology changed, look like round, and aggregated together; In10-12days, UMC and HSF in the MEF feeder grew to like ES cell clones; The cell clones grew more and more bigger during cultiation, their edges were clear, and there were more miscellaneous cells around them.1.6iPS cells clones were picked:We picked up iPS cell clones from UMC and HSF in28-30days after they were infected by virus. These iPS cell clones were respectively named UMC-iPS (C1、C2、C3、C4、C5) and HSF-iPS(C1、C2、C3).1.7The iPS cells were cultured in the feeder or no-feeder cultivation system:iPS cells were in good state during cultivation. Their morphology were spherical shape, looked like the same. The clones gathered together, their edges were clear and tidy. The iPS cells easily occurened differentiation, aging and death, therefore these cells should be handled in time.2Biological identification of iPS cells2.1RT-PCR examing gene expression:①It was three times for expressional level of endogenous genes (Oct4, Sox2, Nanog, Rex1) for the iPS celles than UMC and HSF cells, but the expressional level of endogenous genes were similar for iPS and ES cells.②It is more lower for expressional level of exogenous gene (Sox-2, Klf-4, Oct-4, c-Myc) for the iPS cells than UMC and HSF cells infected with Retrovirus in6days. Among these cell clones. UMC-iPS (C2、C3), HSF-iPS (C1、 C3) were selected to the next biological identification experiment.because their exogenous gene silencing are relatively good than other clones.2.2Identification of gel electrophoresis of exogenous genes:The level of exogenous genes (Oct-4, Sox-2, c-Myc, Klf-4) was high in the iPS cells.but nothing in the UMC and HSF cells. The results showed Oct-4, Sox-2, c-Myc, Klf-4genes already was inserted into the iPS cells.2.3Karyotype analysis:G band karyotype analysis shows that, all iPS cells have46chromosomes, XY type, their cells karyotype were all normal.2.4Alkaline phosphatase staining:①We judged the efficiency of UMC and HSF cell reprogramming with alkaline phosphatase staining method after picked up iPS cell clone. The results shwed that the efficiency of cells reprogramming were respectively1.84%for UMC and0.89%for HSF.②The iPS cells were dyed by alkaline phosphatase staining while they are cultivated in MEF feeder. The iPS cells were dyed purple, the result was positive, feeder and other cells are stained pink or no staining, the result was negative.2.5The expression of ES cell-specific protein:According to results of antigen and antibody immune fluorescence reaction of iPS cell, membrane protein (SSEA3, SSEA4) was stained violet blue, qualitative protein (TRA-1-60,TRA-1-81), nucleoprotein (Nanog) was stained turquoise, all Ips cell dyeing are all positive, iPS cells express ES cells specific proteins.2.6Nanog, Oct4methylation sequencing:Endogenous genes (Nanog, Oct-4genes are) were methylation for UMC and HSF cells and demethylation for iPS cells from the UMC and HSF.2.7Teratoma experiment and biopsy examine:SCID mice grew a teratoma entity in two months after the iPS cells were injected into them. The biopsy showed that it included tissue and cells of endoderm、mesoderm、ectoderm.2.8Embryoid body experiment and RT-PCR examine:iPS cells differiated into embryoid bodies during cultivation in vitro. RT-PCR datas showed that Gene expression level, endoderm gene (AFP, GATA4,SOX17), the mesoderm genes (TBX1), ectodermal genes (PAX6, SOX1), were significantly higher for embryoid body cells than the iPS cells.Conclusions1Generation of human induced pluripotent stem cells from umbilical cord matrix and human skin fibroblasts, in Oct-4, Sox-2, c-Myc, Klf-4as the programming gene,by retroviral mediated transgene technology. 2The iPS cell biology identification displayed the human induced pluripotent stem cells are similar to ES cells in morphology、relative gene expression, Alkaline phosphatase activity,specific protein expression, cell differentiation potential.