| Induced pluripotent stem cells compared to embryonic stem cells have the incomparable superiority, have become an important experimental technology for cell proliferation, differentiation and regeneration medicine researches. Previous researches found that the process of iPSCs generated will experience three morphological changes: type of fibroblast (MEF) to epithelioid cells and then embryonic stem cellclones (iPSCs). Understanding the kinetic changes of the three different cell type during the reprogramming process will help to elucidate the reprogramming mechanism and plays an important role in improving the induced efficiency. Therefore, in this research we established a method of inducing iPSCs without feeder layer, and comparing effect of two different culture medium additives (serum and serum replacement) on iPSCs induction efficiency. Then, we analyse the dynamics characteristics of the PD3, PD6, PD9, PD12 celles during the reprogramming process by using antibody Thy1, SSEA1 and Oct4-GFP reporting system. The results showed that:in serum replacement medium, iPSCs induction efficiency was significantly higher than that in serum culture medium (15.2% vs 2.59%), and also the iPSCs clonal morphology and Oct4-GFP expression was obviously higher in serum replacement medium than that of in serum medium. Furthermore, we used no feeder layer and serum replacement medium as the induction system,staining the celles of PD3, PD6, PD9 and PD12 during the reprogramming process and analyzing by flow cytometry, exploring the kinetic changes of different stages celles specificity markers during the reprogramming process. The results showed that:at PD3, Thy1+SSEAl- cells accounted for 68.6%, Thyl-SSEAl- cells accounted for 23.8%, Thy1-SSEAl+ cells accounted for 6.63%. At PD6, the number of Thyl+SSEA1-cells significantly decreased to 16.8%, and Thyl-SSEAl- cells increased to 55.1%, the number of Thy 1--SSEA1+ cells was significantly improved to 27.3%,so in the early reprogramming stage, the specificity cell markers change as follows:Thy1+SSEAl-cells→Thy1-SSEAl-cells→Thy1-SSEAl+cells. At PD9, SSEAl-GFP+cells accounted for 12.6%, SSEAl-GFP- cells accounted for 18.2%, SSEA1+GFP+cells account for 51.7%. At PD12, SSEAl-GFP+ cells proportion of rised to 23.8%, SSEAl-GFP- cells proportion down to 11.6%, and SSEA1+GFP+cells proportion rised to 57.5%. So, in the late reprogramming stage, the specificity cell markers change as follows:SSEAl+GFP- cells →SSEAl+GFP+ cells. Therefore, the dynamic change of the cell specificity marker during the whole reprogramming process was: Thy1+SSEAl-GFP-→Thy1-SSEAl-GFP-→Thy1-SSEAl+GFP-→Thy1-SSEAl+GFP+. Sorting Thy1- SSEAl+ GFP+cells and culturing on the feeder layer, formed iPSCs clones are in uniform morphology, growth in good condition, clones in neat edge, obvious shading and expression GFP, positive for alkaline phosphatase staining. The immunofluorescence staining analysis shows Oct4, Sox2, Nanog, E-cadherin and SSEA1 were all positive. The iPSCs can differentiation into embryoid bodies in vitro, andexpress three germ layermarker protein,Nestin (ectoderm), Brachyury (mesoderm) and GATA4 (endoderm).Ectopically expressing of transcription factors could reprogram somatic cells into a pluripotent stem cells state and we call this induced pluripotent stem cells (iPSCs). this method initiated anew strategy to establish livestock stem cells for the animal like cattle, sheep, pig, which have difficult in obtaining the embryonic stem cell. Although there have been obtained with partial reprogramed iPSCs, but these iPSCs can not cultured for a long time in vitro, andrely on long-term expression of exogenous transcription factors to maintain the shape of the clone, and endogenous stem cell regulatory networks has not been activated. When the expression of transcription factor was silenced, the iPSCs immediatelydifferentiation, also the cell morphology and pluripotent gene expression have the obvious difference in those iPSCs. The Oct4-GFP reporting system is applied to the iPSCs reprogramming, which provides a powerful technical means to determine whether the endogenous stem cell network is activated or not during reprogramming. In this study, we establish bovine fetal fibroblast cells (TWSIN1-OG) which contain human Oct4-GFP reporter gene, determine the activation of endogenous stem cell network during the process of reprogramming. We used human-derived OCT4, SOX2, KLF4, C-MYC, NANOG, LIN28(OSKMNL) infect TWSIN-OG cells to obtainthe bovine iPSCs.We using different volume of virus infected cells and use the DsRed fluorescent indicating infection efficiency, the results show that, infection rate of 10 μl,20μl,30 μl,40 μl virus was 54.3%,62.2%,74.8%, 79.3%. And then we use of 40μl OSKMNL Lentivirus to induce the bovine iPSCs, observation of cell morphological changes in the process of induction. The results showed that:similar to the mice, at day3, the induced cells began by transformed fibroblast into epithelioid cells, at day6, the epithelioid cells began to gather, at day9, the initial shape of colony formation, at day 12, clone continuegrow and become bigger. From the day 13 to day22, we selected better clones forreplication, but these cells can not long-term stable passage, and the Oct4-GFP also not expression. In the subsequent research work, we will continue to optimized bovine iPSCs induce system, hope to establish a bovine induced pluripotent stem cells which can be long-term cultured in vitro and the stable activation of endogenous stem cell network. |
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