Establishment and characterization of human germ cells derived from embryonic stem cells (hESCs)

Abstract

OBJECTIVE: The ability to generate germ cells from hESCs represents a potential treatment for patients with infertility as well as an essential means of studying the mechanism of germ cell differentiation. The aim of this study is to investigate the feasibility of efficient production of human primordial germ cells (PGCs) from hESCs.DESIGN: In vitro cell culture.MATERIALS AND METHODS: H9 hESCs were co-cultured with MEFs in an optimized media. Cell colonies were stained with germ cell specific antibodies against SSEA-1 and cKit to evaluate for the presence of PGCs by FACS analysis. Populations of SSEA1+/cKit+, and SSEA1-/cKit- cells were sorted and analyzed for mRNA and protein expression of germ cell markers as well as embryonic germ cell (EGC) forming potential.RESULTS: High numbers of SSEA1+/cKit+ PGCs (5-10%) were generated from hESCs after differentiation. Expression of early, migratory, and matured germ cell markers of Stella, Blimp1, Nanog, Dazl, ZP1, ZP3, Acrosin and AMH was detected by qPCR. Protein expression of the pluripotent marker OCT-4 and germline marker VASA was detected by microscopy. Entry into early meiosis was suggested by the detection of of Stra8, Scp1, and Scp3. Microscopy demonstrated nuclear expression of SCP3 in 29% of PGCs. Germ cell and meiotic markers were absent in the SSEA1-/cKit- population. Only SSEA+/cKit+ cells were able to form EGC colonies in vitro. Pluripotential of EGCs was validated by the formation of mature teratomas containing all 3 germ layers in nude mice. PGCs were derived by differentiation of EGCs (∼10%) in vitro after >10 passages at similar efficiency. cKit+/SSEA1+ cells demonstrated imprint erasure characteristic of PGCs in vivo as confirmed by bisulfite sequencing.CONCLUSION: These studies demonstrate that PGCs could be efficiently and consistently generated by differentiation of hESCs without the introduction of reporter genes. The ability to produce PGCs and EGCs is unique and represents an ideal model to study human germ cell biology. OBJECTIVE: The ability to generate germ cells from hESCs represents a potential treatment for patients with infertility as well as an essential means of studying the mechanism of germ cell differentiation. The aim of this study is to investigate the feasibility of efficient production of human primordial germ cells (PGCs) from hESCs. DESIGN: In vitro cell culture. MATERIALS AND METHODS: H9 hESCs were co-cultured with MEFs in an optimized media. Cell colonies were stained with germ cell specific antibodies against SSEA-1 and cKit to evaluate for the presence of PGCs by FACS analysis. Populations of SSEA1+/cKit+, and SSEA1-/cKit- cells were sorted and analyzed for mRNA and protein expression of germ cell markers as well as embryonic germ cell (EGC) forming potential. RESULTS: High numbers of SSEA1+/cKit+ PGCs (5-10%) were generated from hESCs after differentiation. Expression of early, migratory, and matured germ cell markers of Stella, Blimp1, Nanog, Dazl, ZP1, ZP3, Acrosin and AMH was detected by qPCR. Protein expression of the pluripotent marker OCT-4 and germline marker VASA was detected by microscopy. Entry into early meiosis was suggested by the detection of of Stra8, Scp1, and Scp3. Microscopy demonstrated nuclear expression of SCP3 in 29% of PGCs. Germ cell and meiotic markers were absent in the SSEA1-/cKit- population. Only SSEA+/cKit+ cells were able to form EGC colonies in vitro. Pluripotential of EGCs was validated by the formation of mature teratomas containing all 3 germ layers in nude mice. PGCs were derived by differentiation of EGCs (∼10%) in vitro after >10 passages at similar efficiency. cKit+/SSEA1+ cells demonstrated imprint erasure characteristic of PGCs in vivo as confirmed by bisulfite sequencing. CONCLUSION: These studies demonstrate that PGCs could be efficiently and consistently generated by differentiation of hESCs without the introduction of reporter genes. The ability to produce PGCs and EGCs is unique and represents an ideal model to study human germ cell biology.