Isolation, Culture And Identification Of Human Amniotic Mesenchymal Stem Cells

Background and objective:The amniotic membrane is the inner structure surrounding the fetus as it develops in the uterus.At birth it is discarded.This experiment is to investigate the isolation,cultivation,differentiation and identification of human amniotic mesenchymal stem cells(MSCs)in vitro,and to observe the feasibility of using MSCs as seed cells in experimental furthermore.Mesenchymal stem cells(MSCs)are derived from single layer of extraembryonic mesoderm.They are characterized by being multi-potential and capable of self renewal.Thus,they may differentiate into various types of cell.MSCs were initially identified in human postnatal bone marrow(BM).For clinical use,human adult BM is the most common source of MSCs.However,not only the isolation of such stem cells from individuals is invasive and painful but also the expansion potency and differentiation capability decrease with age.In recent years,alternative sources for stem cells have been described including peripheral blood cell,umbilical cord blood cell,muscle cell,and exfoliated deciduous teeth mesenchymal stem cells.But the numbers of cells from these tissues are very limited.Thus,it is meaningful to find new cell replacement without ethic issue,easily accessible and high-yield.We have explored the possibility of a multipotent stem cell residing in the human amniotic membrane. Methods:Placenta was obtained from a normal full-term childbirth,the amnion was separated at aseptic condition and washed with D-hank's,pooled in a dish and cut into the small pieces.And then put the small pieces into the bottle of 25cm~2,the small pieces were cultured in DMEM/F12 containing 10%fetal calf serum.Until the cells reached 80%-90%confluence,then 0.125%trypsin solutin was added and the MSCs were detached from the culture flask walls.The growth cycle and morphological changes of MSCs were monitored by phase contrast microscopy.Growth curves of the cells were drawn with the third passage cells.The MSCs were plated in two 12-well plate and the cell growth was measured by counting method.The specific cell surface antigen of the amniotic-derived MSCs were observed at passage 3～5 by flow cytometry.MSCs were differentiated into adipocytes and osteocytes with the third passage cells,and morphological observations were performed with phase-contrast microscope.Their potential to differentiation into adipocytes and osteocytes was determined by oil red and von kossa staining.Results:After seven days,we could see some cells around the small pieces. When observed,cells displayed in round,spindle,star and polygon shapes.The amniotic-derived cells reached confluence about 80%-90%at about 7d.After passage the amniotic-derived cells achieved complete confluence at 7d-10d.The phenotype of the cultured cells was similar to that of MSCs derived from adult BM.CD29,CD44 and CD105were positive.While CD34,CD45,CDlla, CD1 lb,Pan—CK,CD31 and HLA-DR were negative by Flow cytometry.It can be affirmed as the MSCs which characterized with multi-differentiation potentiality after the lipogenic and osteobtastic differentiation.Conclusions:1.We can get MSCs frorn the amnion,when the little tissue were cultured in DMEM/F 12 containing 10%fetal calf serum. 2.Mesenchymal-like stem cells exist in amnion and have a lower precursor frequency.The cells can differentiate into osteobiast and adipocyte. 3.MSCs in amnion can be isolated and cultivated in vitro,which could be regarded as an alternative source of MSCs for further experimental tests.