Induction Of Pluripotent Stem Cells From Mouse Embryonic Fibroblast Cultures By3Defined Factors

Pluripotent stem cells, which have the potential to differentiate into any of the three germ layers and the ability to self-renew, possess significant application perspective and superiority in cell replacement therapy、gene treatment and developmental biology and so on. At present, the most known pluripotent stem cells are embryonic stem cells, which were generally generated from the inner cell mass of blastocysts. The Pluripotent stem cells also can be obtained by somatic cell nuclear transfer or cell fusion. However, embryonic stem cell research, especially human embryonic stem cells, needs to destroy the embryo or egg cells, thus raises many ethical arguments, and may suffer immune rejection in clinical application and some other problems. The emergence of iPS cells brings the hope to solve these above-mentioned problems. Compared with ESCs, iPS cells have unique advantages:①iPS cells were induced from somatic cells, there were no ethical problems that ESCs faced;②iPS cells had a wide variety of sources, they can be obtained from patients’somatic cells (including lesion somatic cells), to meet the large number requirements in the clinical transplantation treatment by clonal proliferation.③The iPS cells from patients can avoid the problem of immune rejection which had been troubled the regenerative medicine. Therefore, since the appearance of iPS technology, tremendous progress had been made in the study of the induction efficiency, safety and clinical applications. In this study, we introduced3factors(Oct4,Sox2and Klf4) into the mouse embryonic fibroblasts (MEFs) of BALB/C mouse by retrovirus infection and successfully obtained iPS cells derived from MEFs. Then analyzed and identified the expression of stem cell markers and pluripotency of the iPS cells.Objective:1. To generate the induced pluripotent stem cells (iPSCs) from mouse embryonicfibroblast cells (MEFs) by introducing3defined factors, Oct4、Sox2、Klf4.2. To analyze and identify the biology characteristics of the iPS cells, including theexpression of stem cell markers, pluripotency and so on. In order to obtainpluripotent stem cells similar to embryonic stem cells.Methods:1. Preparation of the retrovirus:The retroviral vectors, containing the three genes of Oct4, Sox2and Klf4, PMXs-Oct3、pMXs-Sox2、pMXs-Klf4and Reference plasmid pMXs-EGFP were bought from the Addgene company. Then the four plasmids were amplified by DH5a bacterial, following transfect the four plasmids into the retrovirus packaging cells, Plat-E cells.48hours after transfection, Collected viral supernatants and then used for infection of MEFs after viral supernatants were filtered and concentrated.2. Isolation and culture of mouse embryo fibroblasts:Sterile separated the13.5days pregnancy BALB/C mouse embryos. Digested embryos into single cell suspension and then planted the cell suspension to100-mm tissue culture. The cells within3generations can be used for induction of iPS cells.3. Retroviral infection of MEFs and iPS cells generated:Collected the viral supernatant from the Plat-E dish and then infected MEFs in good condition. Changed the medium every day until the colonies became big enough to be picked up. Colonies should first become visible approximately a week after the retroviral infection. They should become large enough to be picked up around day20.4. Identification of biological Characteristics of iPS cells:Analyzed the morphology, pluripotency gene expression, stem cell surface markers and pluripotency of the iPS cells by Microscopy, alkaline phosphatase staining, reverse transcription PCR (RT-PCR), immunofluorescence assay and teratoma formation.Results:1. Preparation of the retro virus:48hours after the four retroviral vectors transfected into the Plat-E cells, green fluorescent protein can be visible under a fluorescence microscope (pMXs-EGFP), with the transfection efficiency about50-70%. And the Plat-E cells were in good condition.2. Isolation and culture of mouse embryo fibroblasts:The primary MEFs culture adhered3-4h after planted, and the cells were fusiform, polygonal or irregular in shape, in the middle of cells there were1-2round or oval nucleus. The primary MEFs contained lots of hybrid cells, With the increase of passages, the cell purity increased. Generally, relatively pure cells can be obtained after the second generation.3. Retroviral infection of MEFs and iPS cells generated:iPS colonies should first become visible in the microscope approximately a week after the retroviral infection of MEFs. And then the clones continued to proliferate, about12d formed iPS colonies visible to the naked eye. The colonies could grow to large enough when16-20days, and then picked the iPS colonies and planted to feed cells for passaged culture.4. Identification of biological Characteristics of iPS cells:The iPSCs generated from the MEFs showed the typical clone-like growth observed under microscope, round or oval, and had clear boundaries with feeding cells. RT-PCR, alkaline phosphatase staining and immunofluorescence staining demonstrated that iPS cells expressed embryonic stem cell gene and protein. Teratoma formation assays indicated the iPSCs possessed the ability to differentiate into3-germ layers both in vivo and in vitro. As a contrast, the MEFs not infected with the retroviral did not have these above-mentioned ES cell characteristics.Conclusion:1. The iPS cells can be induced by transduction three reprogramming factors (Oct4, Sox2, Klf4) into mouse embryonic fibroblasts.2. The iPS cells, with ES-like biological characteristics, can be long-term passaged and maintained the pluripotency, which means the mouse iPS cell lines are preliminarily established.