Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells

Hsin Fu Chen, Pey Shynan Jan, Hung Chih Kuo, Fang Chun Wu, Chen Wei Lan, Mei Chi Huang, Chung Liang Chien, Hong Nerng Ho

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4-BMP7-BMP8b treatment. Flow cytometry showed that granulosa cell coculture (P < 0.001) or conditioned medium (P = 0.007) treatment for 14 days significantly increased the percentages of differentiated H9 Oct4-EGFP cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both.

Original languageEnglish
Pages (from-to)319-332
Number of pages14
JournalReproductive BioMedicine Online
Volume29
Issue number3
DOIs
Publication statusPublished - Jan 1 2014
Externally publishedYes

Keywords

  • Embryonic stem cells
  • Germ cells
  • Granulosa cells
  • Retinoid acid differentiation

ASJC Scopus subject areas

  • Reproductive Medicine
  • Obstetrics and Gynaecology
  • Developmental Biology

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