An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis

Abstract

Embryoid bodies (EBs) can serve as a system for evaluating pluripotency, cellular differentiation, and tissue morphogenesis. In this study, we use EBs derived from mouse embryonic stem cells (mESCs) and human amniocyte-derived induced pluripotent stem cells (hAdiPSCs) as a model for ovarian granulosa cell (GC) development and steroidogenic cell commitment. We demonstrated that spontaneously differentiated murine EBs (mEBs) and human EBs (hEBs) displayed ovarian GC markers, such as aromatase (CYP19A1), FOXL2, AMHR2, FSHR, and GJA1. Comparative microarray analysis identified both shared and unique gene expression between mEBs and the maturing mouse ovary. Gene sets related to gonadogenesis, lipid metabolism, and ovarian development were significantly overrepresented in EBs. Of the 29 genes, 15 that were differentially regulated in steroidogenic mEBs displayed temporal expression changes between embryonic, postnatal, and mature ovarian tissues by polymerase chain reaction. Importantly, both mEBs and hEBs were capable of gonadotropin-responsive estradiol (E2) synthesis in vitro (217-759 pg/mL). Live fluorescence-activated cell sorting-sorted AMHR2 granulosa-like cells from mEBs continued to produce E2 after purification (15.3 pg/mL) and secreted significantly more E2 than AMHR2 cells (8.6 pg/mL, P < .05). We conclude that spontaneously differentiated EBs of both mESC and hAdiPSC origin can serve as a biologically relevant model for ovarian GC differentiation and steroidogenic cell commitment. These cells should be further investigated for therapeutic uses, such as stem cell-based hormone replacement therapy and in vitro maturation of oocytes.

iPSC-Derived Ovarian Tissue Restores Ovarian Function in Subfertile Mice and After Gonadotoxic Chemotherapy

“…Injection of unsorted differentiated mGriPSCs-GFP into the ovary yielded the highest increase in estradiol production from pre-to post-stem cell injection conditions ( Fig. 3G), consistent with prior observations in which heterotypic differentiated EBs synthesized high levels of steroid hormones [18][19][20]27 . Further, in alignment with our prior studies, the progesterone synthesis between different experimental groups was not significantly different ( Fig.…”

“…The current study builds upon our prior report demonstrating the homotypic differentiation of murine granulosa cell-derived iPSCs into steroidogenic ovarian tissue 18,20 . In the current work, we show that this stem cell-derived ovarian cortex also supports increased generation of differentiating stem cells that express oocyte markers, presumably a larger cohort of primitive oogonia.…”

“…First is the use of granulosa cell-derived iPSCs. To determine whether our endocrine differentiation observations may be extended to other iPSC types, we have previously investigated fibroblast and amniocyte derived iPSCs as well as embryonic stem cells with similar results, although with a lower efficiency 18,20 . We believe the higher efficiency of mGriPSCs in generation of ovarian endocrine tissue relates to the epigenetic memory of these cells.…”

“…After derivation in vitro, mGriPSCs were stably transfected with a green fluorescent protein (GFP) reporter using a lentivirus, followed by differentiation into ovarian tissue as previously described 18,20 (Fig. 2A).…”

“…In this study, we present a novel and exciting approach to the treatment of infertility by generating functional oocytes derived from induced pluripotent stem cells. We previously reported that embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) may be differentiated into steroidogenic ovarian cells that produce physiologic concentrations of the reproductive hormones estrogen and progesterone [18][19][20] . iPSCs generated from mouse granulosa cells (mouse granulosa cell-derived iPSCs, or mGriPSCs) preferentially differentiate into homotypic ovarian cell types due to their epigenetic memory 18 .…”