Effect of BMP4 preceded by retinoic acid and co‐culturing ovarian somatic cells on differentiation of mouse embryonic stem cells into oocyte‐like cells

Abstract

Bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling are the key regulators for germ cell and meiosis induction, respectively. Gonadal tissue also provides an appropriate microenvironment for oocyte differentiation in vivo. The current study aimed to determine whether mimicking in vivo niche is more efficient for oocyte differentiation from embryonic stem (ES) cells. Here, differentiation of mouse ES cells toward oocyte-like cells using embryoid body (EB) and monolayer protocols was induced in the presence (+BMP4) or absence (-BMP4) of BMP4. On day 5, each group was co-cultured with ovarian somatic cells in the presence or absence of RA (+RA or -RA) for an additional 14 days. Our results showed a significant increase in expression of meiotic markers in the +BMP4 condition in EB differentiation protocol. Further differentiation with ovarian somatic cells led to a subpopulation of oocyte-like cell formation. Compared to the controls, the +RA condition resulted in a significant elevation of the meiotic gene expression in contrast to Oct4 that significantly decreased in both protocols. In the cells pre-treated with BMP4 and then exposed to RA in the monolayer differentiation protocol, the gene expression levels of germ cell, Mvh, and maturation markers, Cx37, Zp2, and Gdf9, were also upregulated significantly. Therefore, it can be concluded that +BMP4 and +RA along with ovarian somatic cell co-culture improved the rate of in vitro oocyte differentiation.

In Vitro Generation of Oocyte Like Cells and Their In Vivo Efficacy: How Far We have been Succeeded

“…In cytological studies, co-culturing system can be highly useful to transform many cell lineages. Many researchers have utilized this phenomenon to differentiate various sources-derived stem cells to develop PGCLCs or GCLCs by using MEF co-culture [37,41], granulosa cell co-culture [15,17,22,84,86], placental cell co-culture [12], ovarian stem cell co-culture [20], and fetal gonadal cells co-culture [82]. During whole developmental stages of oogenesis, a developing oocyte is surrounded by different cell layers whose number and shape spontaneously change at different intervals of time.…”

“…In the last few decades, the in vitro development of oocyte-like cells (OLCs) with the ability to cure female reproductive disorders by neo-oogenesis and folliculogenesis has been the main focus of reproductive and stem cell scientists. Researchers have succeeded in deriving primordial germ-like cells (PGCLCs), follicle-like cells (FLCs), and OLCs, as well as in treating animal disease models by using embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and neonatal and adult ovarian stem cell sources from different species including humans [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], mice [17][18][19][20][21][22][23][24], porcine [25][26][27][28][29], bovines [30,31], and goats [32]. Ovaries are the primary source and reservoir of oocytes.…”

“…By using multistep differentiation approach, researchers have used cells that were cultured with specific differentiation mediums and plated onto inactivated mouse embryonic fibroblast (MEF) layers. Furthermore, different source-derived stem cells were either transformed into embryoid bodies (EBs) and/or induced with differentiation promoters along with granulosa cell (GC) co-culturing [12,15,20,22,41]. This approach has resulted in the derivation of OLCs with comparable properties like natural oocytes.…”

“…Induced pluripotent stem cells (iPSCs) have provided a solution to this problem, but these cells also have similar demerits to ESCs. Despite these concerns, OLCs have been developed from ESCs and iPSCs [16,20,24,32,42], and continuous efforts are in progress to eradicate safety issues that are related to the use of ESCs and iPSCs. One such cell source with early developmental cell-like properties is that of very small embryonic-like stem cells (VSELs).…”

Reproductive biology, stem cells biotechnology and regenerative medicine: a 1-day national symposium held at Shahid Sadoughi University of Medical Sciences

“…Bahmanpour presented about mouse ESCs (mESCs) differentiation into oocyte- like cells with ovarian somatic cell co-culturing in combination by bone morphogenic protein 4 (BMP4) and retinoic acid (RA) supplementation ( 13 ). Bahmanpour mentioned in vitro ESCs derived gametes provides powerful model for understanding of epigenetic modifications during gametes development, causes of infertility and other molecular base of reproductive disorders.…”