Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression

Abstract

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

Improved rescue of immature oocytes obtained from conventional gonadotropin stimulation cycles via human induced pluripotent stem cell-derived ovarian support cell co-culture

“…We have previously demonstrated that hiPSC-derived OSCs are predominantly composed of granulosa-like cells and ovarian stroma-like cells. 21 In response to FSH hormonal stimulation in vitro , the OSCs produce growth factors and steroids necessary for paracrine interaction with oocytes and cumulus cells. 21 To investigate whether hiPSC-derived OSCs are functionally capable of promoting human oocyte maturation in vitro , as an approach to rescue immature denuded oocytes, we established a co-culture system of these cells with retrieved denuded immature oocytes and assessed maturation rates after 24-28 hours (Figure 1).…”

“…Human induced pluripotent stem cell (hiPSC) derived OSCs were created according to transcription factor (TF)-directed protocols described previously. 21 OSCs were produced in multiple batches and cryopreserved in vials of 120,000 to 150,000 cells each, and stored in the vapor phase of liquid nitrogen in CryoStor CS10 Cell Freezing Medium (StemCell Technologies).…”

“…Our group has recently demonstrated a novel technology to generate ovarian support cells (OSCs) from human induced pluripotent stem cells (hiPSCs) in a rapid, efficient, and reproducible manner through transcription factor (TF)-directed differentiation. 21 The OSCs are primarily composed of FOXL2+ AMHR2+ granulosa-like cells as well as ovarian NR2F2+ stroma-like cells. These cells are growth factor producing and are steroidogenic in the presence of follicle stimulating hormone (FSH).…”

Human induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturationin vitroafter abbreviated gonadotropin stimulation

“…OSCs were created from human induced pluripotent stem cells (hiPSCs) according to transcription factor (TF)-directed protocols described previously. 41 The OSCs were produced in multiple batches and cryopreserved in vials of 120,000 to 150,000 live cells each and stored in liquid nitrogen in CryoStor CS10 Cell Freezing Medium (StemCell Technologies).…”

“…Our previous work described the generation of ovarian support cells (OSCs) from human induced pluripotent stem cells (hiPSCS) in a rapid, efficient, and reproducible manner through transcription factor (TF)-directed differentiation. 41 The OSCs primarily are composed of FOXL2+ AMHR2+ granulosa-like cells as well as ovarian NR2F2+ stroma-like cells. The OSCs are steroidogenic, producing aromatase in response to follicle stimulating hormone (FSH) stimulation to catalyze the estradiol synthesis pathway, and produce the necessary growth factors needed for robust paracrine interaction with oocytes and cumulus cells.…”