Studies On The Biological Characteristics Of Amniotic Fluid-derived Stem Cells And Their Differentiation Into Hepatocytes

Amniotic fluid is a novel source of stem cells which has attracted intensive attentions in recent years.However,many questions remain uncertain about the methods of isolation and expansion of AFSCs,as well as their characteristics and differentiation potential.In this study,we conducted a set of experiments to acquire systematic knowledge about the properties of AFSCs;meanwhile,to evaluate whether AFSCs could be used for cell-based therapy,we induced them to differentiate into hepatic linage in vitro and in vivo.Our study mainly included three parts of work.1.Isolation,expansion,and biological analysis of human second-trimester AFSCs.In order to compare the effect of different culture conditions on isolation and expansion of AFSCs,we primarily plated the amniotic fluid under 4 conditions: 10%of FBS(fetal bovine serum),20%of FBS,15%of FBS with 4ng/ml of bFGF, and 10%of FBS with gelatin- coating plate.The numbers of primary colonies and the capacity of expansion were compared.The stem cell-like characteristics analyzed included ultrastructure,growth kinetics,immunophenotypes,mRNA expressions,differentiation potential(to adipocyte,osteocyte and neuron) and karyotype analysis.Results:we successfully obtained AFSCs under the condition of 15%of FBS with 4ng/ml of bFGF.AFSCs propagated persistently and rapidly.They shared surface markers of both mesenchymal and embryonic origins.After induced to adipocyte,osteocyte and neuron,they expressed specific markers of targeted cells revealed by RT-PCR or immunofluorescent staining.In addition,AFSCs were non-tumorigenic and kept normal karyotypes both at early and late passages.2.Differentiation of AFSCs into hepatocyte in vitro.This prompted us to design a three-stage strategy to induce AFSCs to differentiate into hepatic linage.The AFSCs were first pretreated with DMSO and EGF for 48 hours;then,they were differentiated into hepatic direction by sequential supplementation of FGF₄ and HGF for total 10 days;at last,they were matured under the functions of HGF,ITS,OSM and dexamethasone.Our results showed that 90%of induced cells presented round-like or polygonal shape;AFP,Alb,CK18,HNF4β,CYPB1 genes were detected by RT-PCR or immunofluorescence.Furthermore,the induced AFSCs could intake and excrete ICG,store glycogen and secrete albumin,meaning that they acquired part of functions of matured hepatocyte.3.Transplantation of AFSCs into CCL4-induced liver injury model of immunodefieient mice.Acute liver injury of immunodeficient mice was induced by intraperitoneal administration of CCL₄.24 hours later,CFDA-SE labeled undifferentiated AFSCs were injected through tail vein.The mice were sacrificed after 3,10,and 20 days of transplantation and liver samples were examined.Results:the labeled AFSCs were found in the mice liver after 3,10,20 days of transplantation under fluorescent microscope.Hepatocyte-differentiated AFSCs were identified by positive staining of human albumin after 10 days of transplantation.The liver of mice receiving AFSCs showed grossly normal histology after 10 days of transplantation;in contrast,pathological changes including infiltration of inflammatory cells and small foci of necrosis were still evident in that of sham-transplanted mice after 20 daysIn summary,AFSCs could be isolated from second-trimester amniotic fluid under appropriate conditions.They harbor potent self-renewal and pluripotent capability while maintaining genetically stable.Their differentiation potential to hepatocyte in vitro and in vivo not only offered a novel method for treatment of liver disease,but also suggested that they might be a promising candidate for tissue engineering and stem cell therapy of other human disorders.