Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells

PLoS One. 2014 Apr 1;9(4):e90454. doi: 10.1371/journal.pone.0090454. eCollection 2014.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Cell Culture Techniques / methods
  • Cell Differentiation* / drug effects
  • Cell Lineage / drug effects
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • DEAD-box RNA Helicases / metabolism
  • Embryoid Bodies / cytology
  • Fibroblast Growth Factor 2 / pharmacology
  • Gene Expression Regulation / drug effects
  • Glial Cell Line-Derived Neurotrophic Factor / pharmacology
  • Human Embryonic Stem Cells / cytology*
  • Human Embryonic Stem Cells / drug effects
  • Human Embryonic Stem Cells / metabolism
  • Humans
  • Leukemia Inhibitory Factor / pharmacology
  • Male
  • Mice
  • Spermatozoa / cytology*
  • Spermatozoa / drug effects
  • Testis / cytology


  • Biomarkers
  • Glial Cell Line-Derived Neurotrophic Factor
  • Leukemia Inhibitory Factor
  • Fibroblast Growth Factor 2
  • DDX4 protein, human
  • DEAD-box RNA Helicases

Grants and funding

This research was supported by the Bio & Medical Technology Development Program (2012M3A9C6049723; PI: D.R.L) of the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology, South Korea. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.