Goat Somatic Cells Reprogramming And Transdifferentiation

Goat pluripotent stem cells include goat embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPS). Goat ESCs or EGCs lines have not yet been established because of unknown appropriate culture conditions in vitro and effect of epigenetic factors with goat pluripotent stem cells. Alternatively, iPS cell technology could provid a new method to generate pluripotent cells. The aim of this study was to select appropriate culture conitions for goat pluripotent cells in vitro utilizing deriving goat pluripotent cells from blastocysts which fertilized and developed in vivo. Also, we tried to induce goat iPS cells from goat embryonic fibroblasts (GEF) with retrovirus encoded by human four factors (Oct4/Sox2/Klf4/c-Myc). At the same time, the reprogrammed somatic cells were transdifferentiated specificly into neural-like cells and oocyte-like cells. The results were obtained as follow:1. Optimized in vitro cultrue condition for goat pluripotent cells25Guanzhong dairy goats were superovulated in total and253embryos were obtained by flushing uterus which included2-cell to16-cell embryos, morulae, blastocysts and hatched blastocysts. The numbers were39,113,94and7respectively. In this study, we obtained10.1embryos from every goat on average. Three different basic media were used which were Knockout DMEM, DMEM and DMEM/F12to detect the effect in sustain growth and proliferation of goat ICM with serum or not. Also, we tried to add small molecular compounds into media to find the effect of proliferation with goat pluripotent cells in vitro. Such as, vitamin C (Vc), Valproic Acid (VPA) and5-Aza-2’-Deoxycytidine (5-AzadC). The result indicated that ICM cells were accelerated the process of attachment and increased the proliferative efficiency cultured in Knockout DMEM with15%fetal bovine serum (FBS). It is also benefit with subculture for goat pluripotent cells in vitro. But goat pluripotent cells differentiated spontaneously with some unknown elements in FBS. We also found that adding small molecular conmpounds coud decrease differentiated spontaneously, but reduce proliferation of pluripotent cells. The goat pluripotent cell colonies were subcultured with mechanical methods up to18passages.2. Induced and reprogrammed from goat fibroblastsIn our study, goat induced pluripotent stem cells (iPS) were reprogrammed from goat embryonic fibroblasts (GEF) with retrovirus encoding human cDNAs of Oct4, Sox2, Klf4and c-Myc. After induced17days in vitro, we obtained goat iPS cells with two kinds of different morphology. One kind of colony was similar with human ESCs in morphology, which was flattened with a neat edge, high nuclear cytoplasmic ratio with one or more nucleolus. The other kind of colony had the characterization of mouse ESCs, which was smooth domed, compact with neat edge and characterised by a high nuclear to cytoplasmic. The goat iPS cells were positive with AP staining and could be cultured and passaged stably in vitro. To detect these goat iPS cells with immunofluorescence, the result indicated goat iPS cells expressed pluripotent transcription factors and stem cell surface antigen, such as Oct4, Sox2, SSEA-1and TRA-1-60, but negative with SSEA-4and TRA-1-81. Because these goat iPS cells could express critical exogenous genes and form embryonic bodies in vitro but without developed teratomas in nude mice, these goat iPS cells still stayed in partially reprogrammed phase.3. Reprogrammed goat fibroblasts transdifferentiated into neural-like and oocyte-like cellsIn this study, goat fibroblasts cells which were infected with retrovirus were transdifferentiated into neural-like cells and oocyte-like cells with RA or5%bovine follicle fluids condition cultue media. After induction for7days and18days, the reprogrammed cells that were stayed in unstable and dedifferentiated stage, and then transdifferentiated into neural-like cells and oocyte-like cells. The result indicated that gene marker which were specific expressed in fibroblasts were down-regulation after infection6days with retrovirus when fibroblasts were in dedifferentiated stage. To detect the neural-like cells and oocyte-like cells with RNA and protein, the result showed oocyte-like cells markers were up-regualtion and β-tublin III was positive in neural-like cells with immunofluorescence. In addition, the oocyte-like cells could be manipulated with micromanipulation that could prove the oocyte-like cells had the similar cell morphology and structer with oocyte.