Coaxing HESC to form Oocyte-Like Structures by Co-Culture with Testicular Extract and Hormones



Jinlian Hua1, 2, *, Bernard E. Tuch , Kuldip S. Sidhu2, 3
College of Veterinary Medicine, Shaanxi Center of Stem Cell Engineering and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China
Stem Cell Group, Diabetes Transplant Unit, Prince of Wales Hospital, Sydney, 2031 Australia
Stem Cell Lab, Faculty of Medicine, School of Psychiatry, University of New South Wales, Sydney, 2052 Australia


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Hua et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the College of Veterinary Medicine, Shaanxi Center of Stem Cell Engineering and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China; Tel: 86-29- 87080068; Fax: 86-29-87080068; E-mail: jlhua2003@126.com


Abstract

Reports have shown that murine embryonic stem cells (mESC) can differentiate into primordial germ cells (PGC) and then to early gametes (oocytes and sperm) that after fertilization form blastocysts. Preliminary studies indicated that hESC also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of hESCs into oocytes. Here, we investigated the effects of human fetal testicular extracts (HFTE) and hormones i.e. follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG) to coax hESC to differentiate into oocytes. The embryoid bodies (EBs) derived from hESC formed ovarian-like structures (OLS) after treatment with HFTE for 7 days and with hormones (FSH + hCG) for 20-30 days. OLS exhibited typical oocyte-like spherical shape of variable sizes and some with zona pellucida-like covering RT-PCR, immunological fluorescence staining and flow cytometry analyses showed that these structures are positive for specific germ cell and oocyte markers such as OCT4, SCP3, C-KIT and DDX4, STELLA, FIGLA, GDF9, NANOG􀉼FSHR, ZP1, ZP2 and ZP3. Quantitative RT-PCR analysis showed that FSHR, GDF9 and FIGLA were up-regulated during OLS differentiation from EB in a time-dependent manner. These results demonstrated that hESC have the ability to differentiate into ovarian structures. This study thus provides an model to study germ-cell formation from hESC and these germ cells may generate a potential source of oocytes for therapeutic cloning.

Keywords: Germ cells , Perimordial germ cell (PGC), embryonic stem cells (ESC) , oocyte , follicle stimulating hormone (FSH), human chorion gonadotropin (hCG ).