TY - JOUR
T1 - Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells
AU - Chen, Hsin Fu
AU - Jan, Pey Shynan
AU - Kuo, Hung Chih
AU - Wu, Fang Chun
AU - Lan, Chen Wei
AU - Huang, Mei Chi
AU - Chien, Chung Liang
AU - Ho, Hong Nerng
PY - 2014/1/1
Y1 - 2014/1/1
N2 - 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.
AB - 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.
KW - Embryonic stem cells
KW - Germ cells
KW - Granulosa cells
KW - Retinoid acid differentiation
UR - http://www.scopus.com/inward/record.url?scp=84964308690&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84964308690&partnerID=8YFLogxK
U2 - 10.1016/j.rbmo.2014.05.009
DO - 10.1016/j.rbmo.2014.05.009
M3 - Article
C2 - 25047539
AN - SCOPUS:84964308690
SN - 1472-6483
VL - 29
SP - 319
EP - 332
JO - Reproductive BioMedicine Online
JF - Reproductive BioMedicine Online
IS - 3
ER -
