Isolation And Identification Of Mongolian Sheep Amniotic Fluid-derived Stem Cells And Its Induction Into Osteoblasts

The aim of this study was to isolate and identify Mongolian sheep amniotic fluid-derived stem cells, and to detect the biological characteristics and differentiation potential of AFSC. This study lay a theoretical foundation for generating the cloned sheep and the transgenic sheep which derived from AFSC.The Mongolian sheep AFSC from different gestation stage were cultured, the result showed that the adhere-rate and activity of cells was greatly affected by the time of amniotic fluid acquisition. The adhere-rate of cells in the mid-gestation was higher than that of early gestation. The activity of cells in the mid-gestation was higher than that of late-gestation. The best time of AFSC acquisition was the mid-gestation.Biological characteristics and configuration of AFSC after a long-term cultivation with 50th or more cell passage sustained stable when 15% FBS added in cell medium.AFSC could be purified within 5th passage by mechanical separation combinated with defined digesting time with trypsin to remove the epithelioid cells from fibrous cells. These cells were shuttle-type, cell confluence directional, and the colony was vortex-like arrangement.The result showed that Oct-4, Nanog, SH2, SSEA-1, CD117 and HLA-A were expressed in different passage AFSC by RT-PCR. AFSC were able to form the embryoid body in vitro, which express marker genes of three embryonic germ layers including fgf5 (ectoderm),α-fetoprotein (endoderm) andζ-globin (mesoderm)。AFSC were induced by dexamethasone,β-glycerophosphate and ascorbic acid. As a result, cells growth was reduced, the edges blurred and multilayer growth and nodules occurred over time. Three weeks after induction, the activity of alkaline phosphatase increased remarkably and Alizarin red staining showed red. These results showed that induction into osteoblasts have been successfully.In summary, these results show that the AFSC obtained in this study have stem cell characteristic, strong proliferation and differentiation potency, and could be induced to differentiate into osteoblasts under certainly condition.