Transdifferentiation Of Human Male Germline Stem Cells To Mature Hepatocytes And Its Signaling Transduction

Objective:Transplantation of human hepatocytes has been regarded as an effective therapy for the end-stage and inherited liver diseases.However,the availability of primary human hepatocytes is rather limited,and these cells are hard to proliferate and maintain their function in vitro.A number of studies have shown that hepatocytes can be derived from embryonic stem（ES）cells,the induced pluripotent stem（iPS）cells,and mesenchymal stem cells（MSCs）.However,there are certain hurdles and unresolved risk before the eventual usage of these stem cells in clinic,e.g.,ethical issues with ES cells,and tumorigenesis and the risk of virus infection associated with the iPS cells.Thus,it is essential to seek a readily available source for cell-based therapy of human hepatocytes.Spermatogonial stem cells（SSCs）are adult stem cells within the testis,and notably,they can acquire pluripotency in vitro and dedifferentiate into ES-like cells which subsequently differentiate into various cell lineages of three germ layers.It has been reported that mouse SSCs could transdifferentiate into functional and mature hepatocytes,reflecting that SSCs have potential application in treating liver diseases.However,the generation of mature and functional hepatocytes from human SSCs has not yet been achieved.There are distinct differences in cell types and phenotype of SSCs between human and mice.In this study,we present a detailed induction protocol as well as molecular and cellular evidence supporting direct transdifferentiation of human SSCs to mature human hepatocytes in vitro and in vivo,which could provide an invaluable and new source of human hepatocytes for the treatment of liver diseases without ethical issues and immune rejection.We also examined the signaling transduction during the transdifferentiation of human SSCs to mature hepatocytes,which sheds a novel insight into molecular mechanisms underlying liver development and SSC reprogramming.Methods:Human SSCs were isolated from OA patients’testes using a two-step enzymatic digestion and differential plating followed by magnetic-activated cell sorting（MACS）.The freshly isolated human male germ cells are phenotypically human SSCs with high viability and purity.To induce the transdifferentiation of human SSCs to hepatic stem cells,we mimicked the condition for liver embryonic development using the conditioned medium containing Activin A,bFGF,Wnt3a,HGF,OSM and DEX.We examined the phenotypic characteristics of different stage cells derived from human SSCs by RT-PCR,immunocytochemistry,and flow cytometry in order to clarify their identities.Thereafter,we measured albumin synthesis,urea production,and indocyanine green（ICG）uptake and release to test whether mature human hepatocytes derived from human SSCs assumed functional attributes of hepatocytes.Meanwhile,we detected the expression changes of several transcripts,e.g.,β-CATENIN,HNF4A,FOXA1 and GATA4,by RT-PCR and/or immunocytochemistry.Furthermore,we probed whether human SSCs could transdifferentiate to hepatocytes in vivo.Mouse models of liver injury were created by carbon tetrachloride（CCl₄）administration.Liver mesenchymal cells were used as the niche inducing human SSC transdifferentiation to hepatic lineage cells.Human SSCs were transplanted with mouse liver mesenchymal cells to the kidney capsule of mice,and the phenotype of the differentiated cells was identified by immunocytochemistry and Western blot.The safety of transplanted human SSCs was also evaluated in important organs of the recipient miceResults:For the in vitro transdifferentiation of human SSC:The freshly isolated human germ cells by MACS expressed specific genes and protein markers of human SSCs,e.g.,UCHL1,GPR125,GFRA1,PLZF,and MAGEA4,suggesting that these cells were indeed human SSCs in phenotype.After 10 days of culture and induction,the morphology of human SSCs was changed and became oval and stereoscopic in shape,which was similar to primary hepatic stem cells.RT-PCR and immunocytochemistry showed that these cells expressed CK7,CK19,CK8,and CK18,rather than TRA1-60,SSEA3 and TRA1-81,reflecting that human SSCs didn’t dedifferentiate into ES-like cells and that they directly transdifferentiated to hepatic stem cells.The high conversion rate was consistent with our data of flow cytometry showing that 81.2%of the cells derived from human SSCs were positive for both CK18 and CK19.After further induction by hepatocyte culture medium（HCM）supplemented with HGF,the cells derived from hepatic stem cells expressed CK8,CK18,ALB,AAT,TAT,CYP1A2 at high levels,whereas CK19 and CK7 mRNA was significantly reduced or undetected in these cells,implicating that these cells possess the characteristics of hepatocytes rather than biliary epithelial cells.We further employed the combination of HGF,OSM and Dex to further facilitate the maturation of hepatocytes.After 10 days of treatment,the cells derived from primary human SSCs had strong mRNA expression of CYP1A2,ALB,and AAT.Significantly,mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes,because they could produce albumin,remove ammonia,and uptake and release indocyanine green（ICG）.These data indicate that human SSCs could convert to phenotypic and functional hepatocytes in vitro.Moreover,expression ofβ-CATENIN,HNF4A,FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes,suggesting this signaling transduction pathway was involved in the conversion of human SSC to mature hepatocytes.For the in vivo transdifferentiation of human SSC:Carbon tetrachloride was effectively used to establish mouse model of liver injury.The isolated liver mesenchymal cells were identified by their morphology and phenotypic markers.RT-PCR showed that Desmin,Emr,v-wf,and Acta2 were detected in the isolated Kupffer cells.Immunocytochemistry revealed the expression of VIMENTIN in the isolated hepatic stellate cells and V-WF in the isolated sinusoidal endothelial cells.These results demonstrated that the isolated cells were phenotypically liver mesenchymal cells.Four weeks after transplantation of human GFP-labelled SSCs and mouse liver mesenchymal cells to the kidney capsule of mice,the cells grew well and they were co-expressing GFP and hepatocytes markers,e.g.,CK18,ALB,AAT and CYP1A2,whereas GPR125 and VASA were undetected in these cells.Furthermore,Western blot showed that ALB,AAT,CYP1A2,AFP,and PCNA were expressed in the transpanted cells.In addition,no obvious leision was observed in the important organs,including hear,kidney,brain,and spleen in the recipient mice.Taken together,these data indicate that human SSCs converted to mature hepatocytes in phenotype in vivo,and transplantation of human SSCs had a good safety,reflecting a great clinic application of human SSCs in treating liver disorders.Conclusion:In summary,we have for the first time demonstrated that human SSCs can directly transdifferentiate into hepatic stem cells that are capable of differentiating into the cells with morphology,phenotype and functional attributes of mature hepatocytes in vitro and in vivo.This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening.We have also revealed thatβ-CATENIN/HNF4A/FOXA1/GATA4 signaling pathway was upregulated during the transdifferentiation of human SSCs to mature hepatocytes,which provides novel insights into mechanisms underlying human liver regeneration and SSC reprogramming.