title: Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.
abstract: The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.
authors.affiliation: Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, Korea.
authors.forename: Jung Jin
authors.initials: JJ
authors.lastname: Lim
authors.affiliation: Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, Korea.
authors.forename: Myung Sun
authors.initials: MS
authors.lastname: Shim
authors.affiliation: Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, Korea.
authors.forename: Jeoung Eun
authors.initials: JE
authors.lastname: Lee
authors.affiliation: Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, Korea; Department of Biomedical Science, College of Life Science, CHA University, Seoul, Korea.
authors.forename: Dong Ryul
authors.initials: DR
authors.lastname: Lee
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D058732:Embryoid Bodies / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D000066449:Human Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008297:Male; D051379:Mice; D013094:Spermatozoa / Q000166:cytology* / Q000187:drug effects; D013737:Testis / Q000166:cytology
chemical_list: D015415:Biomarkers; D051100:Glial Cell Line-Derived Neurotrophic Factor; D053605:Leukemia Inhibitory Factor; D016222:Fibroblast Growth Factor 2; C498288:DDX4 protein, human; D053487:DEAD-box RNA Helicases