In Vitro Chondrogenic Differentiation Of Messenchymal Stem Cells Derived From Human Amniotic Fluid

ObjectiveTo investigate the method of isolation,purification and amplification of messenchymal stem cells(MSCs) derived from second-trimester human amniotic fluid and the possibility of AF-MSCs induced into chondrocyte-like cells in vitro.MethodsFibroblast-like MSCs were separated mechanically from cultured amnioticytes, expanded and analyzed its surface antigens by flow cytometry.The multipotiential marker Oct4 and Nanog was tested by RT-PCR.After 3 passages AF-MSCs were shifted to chondrocyte medium(IMDM supplemented with 10%FBS,1%Amino-max-100,TGF-β1,IGF-1,Dex etc)for 3 weeks.The morphology of induced cells were evaluated by contrast microscopy.The positive cells expressing chondrocyte special extracellular matrix such as CollagenⅡ,Aggrecan,Chondromodulin-1,S100 were confirmed by immunofluorescence. RT-PCR was used to study the mRNA expression of CollagenⅡ,Aggrecan, Chondromodullin-1,S100.ResultsAF-MSCs could be successfully isolated,purified and enriched from the culture of amniocytes using mechanical methods.The expanded MSCs were positive for CD105, CD166,low positive for CD29,but negative for CD34,CD45,that was consistent with the regular characters of MSC.RT-PCR analysis showed the expression of multipotiential marker Oct4,but not for Nanog.After induction by chondrocyte medium for 3 weeks, most AF-MSCs presented typical morphological features of chondrocyte.The immunofluorescence and RT-PCR analysis showed the expression of CollagenⅡ, Aggrecan,Chondromodulin-1,S100 in the induced group. ConclusionsMechanical method is an effective approach to isolate MSCs from second-trimester human amniotic fluid.AF-MSCs can be induced into chondrocyte-like cells after 3 weeks' chondrogenic induction.