Regulation Of Selfrenewal And Differentiation Of Human Embryonic Germ Cells In Vitro

BackgroundIn 1998, human embryonic stem cells (ES cells) and embryonic germ cells (EG cells) were successfully established from inner cell mass (ICM) and primordial germ cells (PGCs) respectively. Thus, their unlimited proliferation and multi-lineage differentiation capacity are critical for delineation of early embryo development, cell therapy and tissue engineering, which are largely based on a fully understanding of the regulatory mechanisms regarding selfrenewal and differentiation of embryonic stem cells in vitro.As long term culture of hEG cells is unavailable, optimizing current culture system stands essential for promoting related research. Up to date, various cytokines have been proven required for in vitro undifferentiated growth of hEG cells, including LIF, bFGF, SCF and Forskolin. Moreover, efforts will be made to establish a safe and efficient supportive feed layer to substitute murine fibroblast STO or MEF, ruling out the xenogenic contamination of human embryonic stem cell cultures. Here, hEG cells are inoculated by coculture of cell mixtures from human 5-9 weeks embryo gonads with LIF-integrated human embryonic lung fibroblasts, and a comprehensive characterization is carried out.Prior to enter gonad ridge contacting intimately with somatic cells to become authentic germ cells, PGCs behave transiently as diploid precursors for germ cells. In the opinion of system biology, germ cells are pivotal due to the equipped genetic information to descendents, while somatic cells ensure a variety of protections and nutrients. Therefore, insights of human embryo ontogeny undoubtedly depends on continuous illustrations of development pathway from PGCs to mature gametes. The finding that in vitro generation of germ cells from ES cells has been listed as top 10 scientific breakout in Science journal, 2004. Actually, the embryonic stem cells/germ cells differentiation platform is not only a good model for reproductive system development but also a novel source for adult germ cells, implicating sufficiently theoretical and practical significance. Particularly as the physiological origin of germ cells, EG cells appear in principle better than ES cells when generation of germ cells is required. In vivo, PGCs can migrate to gonad, amplifying abundantly and initiating meiosis to form germ stem cells. Nevertheless, due to limited samples and absence of specific markers for PGCs, related basic research concerning regulatory mechanisms on PGCs differentiation is rare. In this study, in vitro differentiation of PGCs-derived hEG cells is carefully examined of gene expression profiles, including multipotent stem cell markers Oct4 and Nanog, germ lineage markers Stella and VASA, and meiosis-related molecules SCP1, SCP3, GDF9 and TEKT1.ResultsLIF-transfected human lung fibroblasts as feed layer for human EG cellsLIF, a kind of pleotropic cytokines, is indispensable for maintaining ES cell selfrenewal by suppressing its differentiation. At a first step, the human LIF cDNA was cloned and inserted into the eukaryotic expression vector pcDNA3.0, the pcDNA/lif. Then, embryonic lung fibroblasts (ELFs) with typical fibroblastic morphology as well as high proliferating potential were isolated from lung tissue of human 9-week embryos. Then the ELFs of 5th passages were transfected with pcDNA/lif, and positive clones were collected and expanded after screening with G418 supplement. Stable expression of lif gene was further confirmed by RT-PCR and western blot. Hence, the modified fibroblasts were named hELF/lif.Thereafter, human embryonic gonad cells were transferred to hELF/lif feed layer without addition of recombinant LIF. Upon close observation, PGCs-derived hEG cells were capable of forming canonical nestle-like colonies, which displayed remarkable ALP activity and were stained positive for embryo-specific antibodies (SSEA-1,SSEA-4,TRA-1-60, TRA-1-81), undifferentiated markers (Oct-4,Nanog and Rex-1) and hTERT. Chromosome analysis of 25-day cultures revealed a normal karyotype. Immunohistochemistry and immunofluorescent examination of 14-day EB showed the expression of mesoderm and endoderm markers, Desmin and AFP respectively.RT-PCR analysis of hEG cells, day 7 EB and day 14 EB demonstrated transcriptional activities of the three germ layers: endoderm (α-FP,Pdx-1);mesoderm (CD34,enolase); and ectoderm (Vimentin,NF68), indicating multilineage potential of hEG cells. Of note, hEG cells cocultured with hELF/lif other than with hELF expressed c-myc, the target gene of LIF signaling cascade.In vitro differentiation of hEG cells to germ cells and related molecular mechanismsUndifferentiated hEG cells displayed significant transcription of Oct4 and Nanog, with the latter immediately and markedly downregulated and eventually abolished upon induced to EB. In contrast, expression of Oct4 was retained at a lower level. Within 7-day EB, expression of STELLA and VASA was detected, in consistence with the in vivo finding. Meanwhile, mRNA of oocyte-specific GDF9 and spermatogonium-specific TEKT1 was observed in 3-day and 7-day EB respectively. Thereafter, meiosis marker SCP1 was also detected. Moreover, Nanog could not be detected in SCP3 positive cells within the EB structures. Subsequently, analysis of 7-week and 13-week embryos was performed. Nanog was abundantly expressed by migrating PGCs. Thus, Nanog might function as an inhibitor of embryonic germ cell differentiation. As decreased expression of Nanog is indispensable for meiosis in vivo, downregulation of Oct4-modulated Nanog appears also critical for hEG cell differentiation in vitro. However, direct evidence is required in the future.ConclusionsHere, we have shown that genetically modified hELF could express exogenous LIF and acted as a novel and appropriate niche for in vitro growth of hEG cells, reflecting promising utilities in further basic research and clinical cell therapies. Furthermore, hEG cells are also capable of being driven into the germ cell pathway as evidenced by a well orchestrated gene expression, highlighting its great potential either as an in vitro model mimicking germ cell ontogeny or as a valuable pool for germ cell preparations to a great deal of patients.