Isolation And Culture Of Goat ES-Like Cells And ES Detrived Epithelial-Like Cells

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the mammalian blastocyst or primordial germ cells. They are able to undergo self-renewing cell division under specific cell culture conditions for extended periods, thereby maintaining their pluripotency. Therefore, those characteristics provide an efficient and broadly applicable approach to generate cloning animals, transgenic animals and animal mammary gland bioreactor. However, to date except for a few mouse and human ES cells lines were successfully established, others were not. Research on goat ES cell lines has not been reported.In addition, ESCs can also differentiate into a variety of different cell types when cultured in vitro. Then, a great deal of ESCs research has focused on directed differentiation for decades. To date, many types of cells including myocardial cell, hematopoietic stem cells, neural cells, endothelial cell and epithelial cells have been derived from ES cells. It was reported that ES cell can further differentiate into epithelial-like cells under special conditions. In addition, some studies reported that, gene-modified ES-derived epithelial-like cells can survive for a long time in vivo and expressed foreign gene after they were transplanted into recipient breast tissue, which provides us a new method to produce animal mammary gland bioreactor.This study aimed to obtain better goat feeder layer cell, screen culture and cell factors for ES-like cells’growth and long-term culture from Wuhan domestic goat. We also establish a method for ES-derived epithelial-like cells culture, which can supply cell source for further generating animal mammary gland bioreactor by transplanting genetically modified stem/progenitor cell. The main results as follows:1. In order to prepare feeder layer cells for isolating local goat ES-like cells, goat fetal fibroblasts (GFFs) were isolated by attaching tissue explants from day-45 to 60 goat fetus and purified by trypsin, the cells could be subcultured to 20 passage and had normal phenotype and excellent viability;2. Three types of cells:①mouse embryonic fibroblast (MEFs);②goat embryonic fibroblast (GEFs);③C2C12 (mouse myoblast cell line), were as feeder layer cells for isolating local goat ES-like cells. The result showed that:C2C12 can promote adherence (100%), better than MEF (81.8%) and GEF (88.5%), but not benefit for restraining differentiation state, and merely obtained the 3 passage ES-like cells. 3. Three methods for isolating goat ES-like cells:①whole embryo cultured:after develop to incubated blastula, divided embryo in ordinary culture, we use this condition for each future passage;②whole embryo cultured:after develop to incubated blastula, divided embryo in 10 X LIF and SCF culture, we use this condition for each future passage;③after merged in enzyme for 5～8 min, the acquired embryo was divided by glass needle directly. The results showed that division of inner cell mass with high concentration of LIF and SCF cell factors could help the cells stay in undifferential condition (8 passages) after cultured embryo to incubated blastula stage.4. Three culture media:①the media of DMEM including 20% FBS,20ng/mL LIF, 10ng/mL SCF and 0.1mmol/Lβ-Me;②the media of DMEM including 20% FBS, 20ng/mL LIF, lOng/mL SCF, 0.lmmol/Lβ-Me and 20μg/mL insulin;③the media of DMEM including 20% FBS,20ng/mL LIF, 10ng/mL SCF, 0.1mmol/Lβ-Me,20μg/mL insulin and lOng/mL heparin. The result showed that:the media containing both heparin and insulin (9 passages) sustain longer period of life than only added LIF and SCF (4 passages).5. The goat ESCs-like were isolated from goats embryos had the typical type of ES-like in ES morphology. The nuclear type performance for diploid karyotype analysis was normal. Most goat ES-like colonies were AKP positive and immuno-cytochemically characterized with positive Oct-4 and SSEA-1 staining. ESCs specific transcription factor Oct-4 and Nanog were detected in those cells by RT-PCR method. In vitro, those cells could differentiate into epithelial-like cells, vacuole-like cells and cardiomyocytes.6. To initiate differentiation, goat ES-like cell colonies (3-4 passages) were dispersed to small clumps, allowed to grow on mitotically inactivated GEF feeder cells. For the additional 20d of culture, the epithelial-like cells appeared, and obtained the 11 passages the epithelial-like cells. The nuclear type performance for diploid karyotype analysis was normal. Those cells can express specific genetic markers of epithelial cells including K10, K18 and K14 by RT-PCR method.