In Vitro Culture And Induced Differentiation Of Goat Fetal Lung-Derived Mesenchymal Stem Cell

Mesenchymal stem cells (mesenchymal stem cells, MSCs) is a kind of stem cell, which have the characteristic of strong self-renewal and pluripotency. MSCs occur in variety of tissues of animal fetuses and adults, existing researches have been extracted that there have been isolated mesenchymal stem cells from various tissues of multiple species, the most studied MSCs is bone marrow-derived MSCs, and there is still very rarely reported about the research of the source of fetal lung stem cells. Compared to bone marrow-derived MSCs,fetal lung mesenchymal stem cells have the advantages of easily obtained, numbers of cells, easily separated obtained and in vitro amplified and so on, so creating a mammalian fetal lung-derived stem cells with being isolated and cultured and identification methods can enrich source for stem cells, which also have catalytic role in the study of cell development and differentiation and the establishment of disease models.In this study, to the Alps cashmere goat fetal lung tissue as material source, we first isolated of mesenchymal stem cells which can long-term subculture in vitro,then identified the cells morphology, growth performance, marking the identification of genes and other aspects, last,we induced mesenchymal stem cells into adipogenic, osteogenic,chondrogenic,islet and neural cells to verify the pluripotent.In this study, we use two-step enzymatic digestion and differential adhesion wall to separation Alps cashmere goat fetal lung tissue, by morphological observation, we found isolated the goat fetal lung cells were highly homogeneous, showing a fibroblast-like cell morphology, strong proliferation, when spread to more than20generations, in vitro passage cultured cells still maintain good biological characteristics. Further identify the characteristics of MSCs by karyotype analysis, growth curve, immunocytochemistry, RT-PCR technology. The results showed that the isolated of goat fetal lung cells have a normal diploid karyotype (2n=60); growth of normal in vitro and keeping stable biological characteristics; RT-PCR display positive expression of mesenchymal stem cell marker gene CD44; expression the immunocytochemical detection cell surface markers gene CD13, CD29, CD44, CD90, CD106and negative in control group, negative of hematopoietic stem cell marker gene CD45. The results showed that the isolated cells have the biological characteristics of goat mesenchymal stem cells.On the basis of determine a suitable culture and identification methods in the goat fetal lung mesenchymal stem cells (LMSCs). Further more,we induced goat fetal lung mesenchymal stem cells into fat, osteogenic, chondrogenic, islet (3cell and neurons cells to observe the case of differentiation pluripotency to the three germ layers of goat fetal lung mesenchymal stem cells. After cell staining, immunocytochemistry and RT-PCR identified, the results show:adipogenic cells were treated with oil red-O staining apparent lipid droplets, RT-PCR display the adipogenic cell-specific gene by peroxisome proliferator activation receptor expression was positive; osteogenic by AgNO3staining obvious calcification substances, RT-PCR display osteoblast-specific gene expression of collagen I positive; blue cartilage induced by Alcian blue staining, RT-PCR display cartilage specific genes COL2A1expression positive; into islet beta cells by dithizone staining, the cell mass, and was scarlet, RT-PCR display islet βcell marker genes Pdxl expression positive; into nerve cells by immunocytochemistry testing and found that the cytoplast was found red in positive characteristics and the neuron--specific gene NSE expressed positively. These results suggest that the separation of the goat fetal lung MSCs can be successfully differented into fat, bone, cartilage, nerve cells and pancreatic β cell, differentiation potential to develop into cells of the three germ layers.In summary, the isolated of goat fetal lung cells in this study have the biological characteristics of mesenchymal stem cells and pluripotent, which confirm that we have successfully established a complete set of isolation and purifiedand identification system of goat fetal lung mesenchymal stem cells, we have got the goat fetal lung mesenchymal stem cells.