Identification of Stem Cell-Like Cells in the Ovary

Abstract

Understanding the function of stem cells and cellular microenvironments in in vitro oogenesis, including ovarian folliculogenesis, is crucial for reproductive biology. Because mammalian females cannot generate oocytes after birth, the number of oocyte decreases with the progression of reproductive age. Meanwhile, there is an emerging need for the neogenesis of female germ cells to treat the increasing infertility-related issues in cancer survivors. The concept of oocytes neogenesis came from the promising results of stem cells in reproductive medicine. The stem cells that generate oocytes are defined as stem cell-like cells in the ovary (OSCs). Several recent studies have focused on the origin, isolation, and characteristic of OSCs and the differentiation of OSCs into oocytes, ovarian follicles and granulosa cells. Hence, in this review, we focus on the experimental trends in OSC research and discuss the methods of OSC isolation. We further summarized the characteristics of OSCs and discuss the markers used to identify OSCs differentiated from various cell sources. We believe that this review will be beneficial for advancing the research and clinical applications of OSCs.

Introduction

The central dogma of reproductive biology states that while mammalian males can produce sperms throughout their lifetime, mammalian females are born with a limited number of oocytes. The follicles containing oocytes are the basic unit of female reproduction. However, the number of follicles that mature into oocytes and the number of mature follicles that acquire dominance decline with reproductive age. The alterations in follicular development are the critical physiological factors limiting the reproductive span of females. Female germ cells identified during gestation migrate to the gonadal ridge and proliferate. These germ cells develop into the primordial follicles, which subsequently develop into functional follicles [1]. However, besides this well-established physiological process, studies have shown that some mammals, including humans and rodents, possess active ovarian stem cells (OSCs) to generate new functional oocytes [2–5].

Several studies have reported the existence and origin of OSCs and derived-OSCs from diverse sources, including bone marrow, ovarian cortex, ovarian epithelium, skin-derived stem cells, and pancreatic stem cells [6–13]. The OSCs can proliferate and differentiate into various developmental stages of oogenesis. Further, the OSCs or ovarian stem-like cells have been analyzed using several methods, including fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS) [4, 6–8, 13–21], and differential adhesion- and morphology-based methods [22].

As stated earlier, the OSCs can generated new oocytes [7, 23]. However, due to the methodological problem of obtaining OSCs from the ovary, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are used to generate new oocytes in vitro [24–26]. As shown in Fig. 1, OSCs may generate new oocytes through the processes of folliculogenesis and oogenesis.

Fig. 1.

Applications of OSCs. Studies on OSCs can reveal the mechanism of oocyte development and advance the understanding of the female reproductive system. OSCs can be used to preserve oocyte for women who lose the ovarian reserve due to aging or malignancy, to treat female infertility, and to produce new eggs

Hence, in this review, we describe the origin and characterization of OSCs and discuss their differentiation into oocytes, ovarian follicle cells, and granulosa cells. Moreover, we provide information about the overall trends in OSC research and its future potential. Untapping the vast potential of OSCs to form oocytes in can contribute to the treatment of infertility in cancer survivors, treatment of premature ovarian insufficiency (POI), and help preserve fertility in aged women.

Experimental evidence of OSC

As stated earlier, it was thought that mammalian females could not produce new oocytes after birth. Hence, early reports on the presence of OSCs started a debate about female oogenesis [27]. The dogma that mammalian females have finite oocytes throughout their lifetime prevailed until 2004. In 2004, Tilly and coworkers revealed the existence of proliferating germ cells, which can develop into new oocytes and follicles in the ovaries of cleared mice [5]. Subsequently, numerous studies have shown the existence of OSCs in mice [6, 19, 28, 29], humans [7, 8], and other mammals. These studies raised several questions, including why women undergo menopause if OSCs exist [30]. Although these controversies are ongoing (Table 1), we may still use OSCs to resolve menopause-associated problems.

Table 1.

Debate on the existence of OSCs

Detected markers	Not-detected markers	Species	Results	Methods	References
Existence
SSEA-4, FRAGILIS, DPPA3GAPDH	GDF-9, SYCP 3(small immature oocytes)	Human	Oocytes differentiated from ovarian cortex in menopausal women	DEP array technology	[7]
				Anti-DDX4 antibody cell sorting
				Auto MACS
				Dd (droplet digital) PCR
SSEA-4, OCT4, SOX-2, NANOG, LIN28, STELLADDX4/VASA, MCAM/CD146, Thy-1/CD90, STRO-1		Human	Pluripotent/multipotent stem cell differentiated from ovarian cortex in human	FACS	[48]
				qPCR
				Western blotting
				Immunohistochemistry
OCT4, MVH, DAZL, STELLA, FRAGILIS, NOBOX	SCP3, HDAC6, GDF9, ZP3, SSEA1	Female mice	BM as a source of germ cells that generate oocytes	FACS	[6]
				RT-PCR
		Human		Immunohistochemistry
DDX4, DAZL, STRA8, DMC1, PRDM1, SCP3, OCT-4, SOX2, NANOG, SSEM1		Mice	Germline and pluripotent stem cells that differentiated into oocytes and somatic cells in adult mouse	RT-PCR	[49]
				TEM analysis
				Western blot analysis
Non-existence
	GDF9, DDX4, ZP3, SYCP3	Mice	No GSCs producing oocytes in adult mammal ovary and not anymore reproduce oocytes after fetus	DT-mediated oocyte ablation (eliminating the existing oocytes)	[50]
				RT-PCR
	GFP	Mice	No evidence of oocyte and follicle regeneration	DAPI staining	[51]
				Anti-GFP and anti-MVH antibody techniques
				Immunohistochemistry
	SOX2, OCT4, STELLA, DDX4	Mice	No postnatal follicular renewal	RT-PCR	[32]
			No mitotically active DDX4-expressing FGSC in mouse ovary	Live cell imaging
VASA	SCP3, PRDM9, SCP1, SPO11, C-KIT, TERT, NOBOX, KI-67, PCNA	Adult human ovaries	No existence of active meiosis, neo-oogenesis and GSCs in normal adult human ovaries	RT-PCR	[31]
				Western blotting
				Immunohistochemistry

Molecular analyses have identified specific markers of OSCs. For example, in one study, a fluorescence-tagged dead box polypeptide 4 (DDX4), also known as mouse vasa homolog (MVH), was used as a specific marker of mouse germ cells. Moreover, GFP-tagged OCT4 can revealed the differentiation of OSCs into various stages of oogenesis [30]. Further, meiotic markers can mark OSCs since isolated OSCs can differentiate into germ cells through meiosis. In fact, using meiotic markers SCP3, SPO11, and DMC1 in mice, Tilly et al. identified OSCs that undergo postnatal oogenesis. They also showed that the ZP family protein markers could identify these OSCs [30]. Apart from these, several other studies have identified various markers related to oogenesis. However, a study revealed that DDX4 was not exclusively expressed in the ovary, and thus isolated cells expressing DDX4 may not only represent germ cells [34].

Contrary to the above reports, some studies did not find evidence of OSCs and therefore proposed that ovarian germline stem cells do not exist in normal adult human and mouse ovaries [31, 32]. A previous report also showed that new follicles were not generated after depletion of the primordial follicles, which is the main reserve of oocytes in the mouse ovaries [33].

Sources, isolation and characterization of OSC

Several studies have demonstrated that isolated OSCs can be induced to form new oocytes (Table 2). Ovarian stem-like cells have been isolated from various tissues including bone marrow [6], ovarian cortex [7], ovarian surface epithelium (OSE) [8, 9], skin-derived stem cells [10], and pancreatic stem cells [12], using MACS and FACS. Alternatively, ESCs and iPSCs have also been used to generate oocytes. Figure 2 shows the potential sources and isolation methods of OSCs.

Table 2.

Isolation of OSCs from various sources

Origin	Markers	Species	Isolation methods	References
Bone marrow	OCT4, MVH, DAZL, STELLA, FRAGILIS, NOBOX	Mouse	FACS	[6]
Ovarian cortex	GDF-9, SYCP3, DPPA3, GAPDH SSEA-4, FRAGILIS	Human	DEPArray technology	[7]
			Anti-DDX4 antibody cell sorting
			autoMACS
Skin-derived stem cell	OCT4, GDP9B, DAZL, VASA, ZPB, ZPC	Porcine fetus	RT-PCR	[10]
			Western blotting
			FACS
Pancreatic stem cell	OCT4, SSEA-1, SCP3, DMC1, VASA, GDF-9	Rat	RT-PCR	[12]
			Immunocytochemistry
Ovarian surface epithelium	ZP3, SCP3, C-KIT	Human	MACS	[8]
			FACS
			Immunocytochemistry
			Genetic analyses
Mouse ovary,	NOBOX, GDF9, ZP1-ZP3	Mouse	FACS	[13]
Human ovarian cortex	PRDM1, DPPA3, IFITM3, TERT	Human
Ovarian surface epithelium	SSEA-4, OCT4, FRAGILIS, STELLA	Human	Immunomagnetic sorting using SSEA-4	[11]
	DAZL, VASA, GDF-9, SCP-3, NANOG, CD133	Sheep

Fig. 2.

Isolation of OSCs. OSCs can be obtained from mammalian bone marrow, ovarian cortex, and ovarian surface epithelium. FACS or MACS are used to isolate OSCs from these sources. Isolated OSCs can be differentiate into mature oocytes

The expression of germline markers in bone marrow cells and bone marrow transplantation renews oocyte production in wild-type mice sterilized by chemotherapy [6]. OSCs can also be isolated from the ovarian cortex of both non-menopausal and menopausal mammals using anti-DDX4 antibody-based cell sorting [7, 13]. In one study, DDX4-positive cells were enriched by the autoMACS separator and expressed germline cell markers, including SSEA-4 and FRAGILIS [7]. Furthermore, the small SSEA-4 positive cells isolated from OSE by MACS and FACS were found to express primordial germ cell markers (PRDM1, PRDM14, and DPPA3), pluripotency markers (OCT4, SOX2, SSEA-4, SALL4, CDH1, and LEFTY1), and oocyte-specific markers (ZP3, SCP3, and c-KIT) [8]. Further, OSE can differentiate into oocytes and granulosa cells [9]. Moreover, very small embryonic-like (VSEL) stem cells and oocyte-like cells (OLCs) obtained from OSE scrapings can proliferate and differentiate into oocyte-like cells [11].

Skin-derived stem cells can be a source of OLCs. A study showed that OLCs derived from porcine skin-derived stem cells are defective in entering meiosis [10]. Further, according to Danner et al., adult pancreatic stem cells can differentiate into cell types of all three germ layers in vitro. It was shown that adult pancreatic stem cells could differentiate into OLCs that form follicle-like structure and express typical germ cell markers [12]. Hence, based on the above reports, we deduced that the existence of OSCs. However, extensive investigation is needed to unravel more sources of OSCs in vivo.

Markers are specific transcription factors, cell-surface, or cytoplasmic proteins expressed exclusively and in a time-dependent manner during in vivo development and differentiation. We obtained information on marker proteins of gern cells from in vitro developmental studies (Table 3). Pluripotency markers, such as OCT4, SSEA-4, CD133, FRAGILIS, and STELLA, mark the primordial germ cells. Furthermore, germ cell markers DAZL and VASA are expressed in VSELs stem cells and ovarian germ stem cells [11]. The OLCs, derived from the VSEL stem cells and ovarian germ stem cells, express germ cell markers, such as DAZL, GDF9, VASA, SCP3, and OCT4. During the differentiation of OSCs into OLCs, germ cell cysts/nests, mitochondrial aggregates, and cytoplasmic streaming are observed [11].

Table 3.

Characterization methods of  OSCs

Culture media	Specific markers	Species	Size	Limitations	Characteristics	Methods	References
DMEM/F12	SSEA-4, OCT4 FRAGILIS, STELLA, DAZL, VASA, GDF-9, SCP-3, NANOG, CD133	Human	VSEL (1–3 μm in size)	Stem cells exhibit germ cell cysts, Balbiani body-like structure, cytoplasmic streaming	Spherical oocyte like structures exhibit cytoplasmic streaming or rotational movement	RT-PCR, Immunomagnetic sorting using SSEA-4, H&E staining, Western blotting, Immunohistochemistry	[11]
		Sheep	OGSC (4–7 μm in size)
	MVH, C-KIT, DAZL, DPPA3, PRDM1, FRAGILIS, OCT4, SOX2, NANOG, TERT	Mice	10–20 μm in diameter	Oogenesis delay menopause and cure ovarian dysfunction	Isolation procedures have no mediators of membrane binding (antibodies and magnetic beads)	RT-PCR, Strategy of Differential adherence selection	[28]
	OCT4, SSEA-4,, NANOG, SOX-2, TERT, STAT-3, C-KIT, DAZL, GDF-9, VASA, ZP3	Menopausal woman, Rabbit, Sheep, Marmoset monkey	1 ~ 3 μm PSCs and 4 ~ 7 μm PSCs		It was indicated prominent nucleus and peri-nuclear accumulation of organelles	RT-PCR, H&E staining	[3]
DMEM	OCT4, VASA, C-KIT, NANOG, SOX2, SSEA1, CYCLIND1, DAZL, PLZF,	OCT4-GFP transgenic mice			Rapamycin promote proliferation, inhibit differentiation of mouse FGSC	qRT-PCR, Immunohistochemistry	[34]
	VASA, DAZL, NANOG, OCT4	Chinese soft-shell turtle	PSO1 10 $\mathrm{\mu }$ m in diameter, PSO1 cells may be germline stem cells	Fertility problems and the long term cryopreservation	Round shape and large nuclei containing 1-2prominent nucleoli	RT-PCR, Immunohistochemistry	[35]
					Most of the thecal cells had vanished after 10 passages, and the majority of the remaining cells were germ cells
	OCT4, DPPA3 FRAGILIS MVH, DAZL PRDM1, DMC1 SYCP1, YBX2 ZP3, GDF9	Six week old mice		Infertility and ovarian aging	AKT pathway may regulate FGSC self-renewal	Differential adhesion method, RT-PCR, Immunohistochemistry	[29]
	GDF-9, SYCP3 DPPA3, GAPDH SSEA-4, FRAGILIS	Human	80 ~ 90 μm in diameter of OLCs	The rate of differentiation of OSCs was low	OLCs expressed oocyte terminal differentiation genes, GDF-9 and SYCP3 in menopausal women	DEPArray technology, Anti-DDX4 antibody cell sorting using FACS, MACS	[7]

While previous experiments used FACS or MACS to isolate female germline stem cells (FGSCs), they can also be isolated using a differential adhesion-based method (antibody-coated beads) that does not rely on binding to a specific surface marker. Furthermore, germline markers (MVH, DAZL, DPPA3, PRDM1, and FRAGILIS) and pluripotency markers (OCT4 and TERT) expressed in string FGSCs are similar to FGSCs [28]. Remarkably, few DA-FGSCs could differentiate into OLCs that express ZP3 and GDF9. These cells can further differentiate into functional eggs [29].

A study using OCT4-GFP tracing found that FGSCs express pluripotency, germline, and proliferation-related markers. They further demonstrated that FGSCs are similar to spermatogonial stem cells (SSCs) and ESCs, and can differentiate into all three germ layers [34]. Another study isolated and cultured ovarian stem cell-like lines (PSO1) from the Chinese soft-shell turtle (Pelodiscus sinensis). PSO1 cells express the germline markers VASA and DAZL and the pluripotency gene NANOG. Furthermore, PSO1 cells are highly proliferative due to high levels of proliferating cell nuclear antigen. Thus, the characteristics of PSO1 cells are similar to those of germline stem cells [35].

The characteristics of OLCs derived from OSEs have been analyzed experimentally. OSE from rabbit, monkey, sheep, and menopausal women can form oocyte-like structures, embryonic stem-like cells, and embryoid bodies (EBs). Further, oocyte-like structures derived from human and sheep OSE express germ cell-specific markers (c-KIT, DAZL, GDF-9, VASA, ZP3, and OCT4) and formed germinal vesicles, a distinct zona pellucida, and extrusion of the polar body [3].

Differentiation of stem cells into oocytes

Currently, oocytes are derived from differentiated OSCs, ESCs, and iPSCs. Female ESCs and iPSCs can differentiate into primordial germ cell-like cells and further develop into reconstituted ovaries in vitro (Table 4). Remarkably, the primordial germ cell-like cells have the potential to form germinal vesicle-stage oocytes that generate fertile offspring after in vitro maturation and fertilization [19]. In addition, oocytes can be derived from differentiated SSCs. The cells derived from isolated SSCs express oocyte-specific genes (GDF-9, NOBOX, OOGENESIN, H1FOO, ZP3, GDF-9, and SCP3). Therefore, SSCs are thought to differentiate into OLCs. The SSCs-derived oocytes are of the XO karyotype since PCR analysis of the SRY gene showed that the YO cells died during growth [36]. Further, ESCs can differentiate into oocytes that undergo meiosis and develop into follicle-like and blastocyst-like structures [15].

Table 4.

Differentiation of stem cells into oocyte or oocyte-like cells

Culture	Origin	Species	Markers	Methods	References
IVD culture	PGCLC	Mouse	DPPA3, BMP15, SOHLH1, NOBOX, ZP1, NPM2, STRA8, GDF9, SOX2, PRDM14,	FACS, RNA-seq analysis, Chimaera analysis, COBRA analysis, Immunohistochemistry	[14]
IVG culture
IVM culture	PGCLC	Mice	DDX4, SYCP3, DAPI, BVSC	MACS	[19]
Culture including KO-DMEM medium	Mouse male Spermatogonial stem cell -PSCs	C57BL/6 transgenic mouse	OCT4, GDF9, ZP3, SCP3, STELLA MVH, GAPDH	MACS, RT-PCR	[36]
Culture with mitotically-inactive mouse embryonic fibroblasts (MEFs)	Adult mouse ovary	Human	DDX4, PRDM1, DPPA3, IFITM3, YBX2, MSY2, CONTRIN, KIT, LHX8	FACS, Immunohistochemistry	[4]
	Human ovarian cortical tissue	Mouse
Culture including DMEM	Bone marrow	Mouse	OCT4, MVH, DAZL, STELLA, FRAGILIS, NOBOX, L7	FACS	[6]
	Peripheral blood
	Germ cell purified from female mice	Mice	GFP, MVH, DAPI, OCT4, SSEA-1	MACS, FACS	[16]
	Cultured embryonic stem cell	Mouse	OCT4-GFP, VASA, C-KIT, DMC1, SCP3, OCT4, Β-Actin, GDF-9, ZP1, ZP2, ZP3, FIGΑ, HAND1, PI-1, MASH2, Tpbp	FACS, RT-PCR, Immunohistochemistry	[15]
DMEM/F-12	Woman’s ovarian cortex	Human	GDF-9, SYCP3, DPPA3, GAPDH SSEA-4, FRAGILIS	DEPArray technology, Anti-DDX4 antibody cell sorting, FACS	[7]
Long term culture	Marmoset ovary	Marmosetmonkey	VASA, DAZL, NOBOX, DPPA3, SALL4, LIN28, OCT4A, SCP3	MACS, RT-qPCR	[20]
Culture in medium containing follicular fluid and fetal bovine serum	Skin-derived stem cell	Mouse	FRAGILIS, NANOG, STELLA, GDF9B, OCT4, SOX2, ZP1, ZP2, ZP3, DAZL, VASA	Flow cytometric analysis, RT-PCR, Immunohistochemistry	[37]
Gelatin-coated culture	Ovarian tissue	5–7 day old mice	OCT4, SSEA1, DAZL,C-KIT, NANOG, MVH, GDF9, SCP3, FRAGILIS	Preplating technique, RT-PCR, Immunohistochemistry, Alkaline phosphatase activity	[22]
Culture onto a GC monolayer	Ovarian cortical tissue	Female mice, 26 ~ 43 years old women	DDX4, DAPI, OCT4, GAPDH, IFITM3, DAZL, STELLA, DPPA3, C-KIT, BLIMP-1, STRA8, SYCP3, FIGLA, GJA4, ZP1-3, REX-1, NANOG, SOX2	MACS, RT-PCR, Immunofluorescence analysis, Time-lapse analysis	[21]
Culture consisted MEM-α	Bovine ovarian stem cells	Adult mixed breed cows	GAPDH, SOX2, OCT4, VASA, DAZL, ZPA, GDF9, SCP3	RT-PCR, Immunohistochemistry	[38]

Germ cells can differentiate into oocytes under appropriate conditions. Germ cells that aggregate with fetal gonad somatic cells can differentiate into mature oocytes [16]. OLCs derived from the culture of marmoset monkey OSE express germ cell markers (VASA, DAZL, NOBOX, DPPA3, and SALL4) and the meiotic marker SCP3. These OLCs, which are floating spherical cells, are 20–40 μm in diameter and develop into germline cells [20].

In addition, OSCs were isolated from human ovarian cortical tissues and mouse ovaries using a FACS-based approach using the DDX4 antibody. These OSCs expressed primitive germline markers (PRMD1, DPPA3, and IFITM3). GFP-expressing mouse OSCs were injected into the ovaries, and developing follicles with GFP-positive oocytes were detected. Furthermore, oocytes enclosed within somatic cells have been generated from GFP-expressing human OSCs through viral transduction [4].

GFP-positive skin-derived stem cells isolated from OCT4-GFP transgenic mice differentiated into OLCs that were 40–45 μm in diameter and expressed oocyte markers (OCT4, GDF9B, VASA, DAZL, and FIGα) in vitro. In addition, aggregates of skin-derived stem cells and newborn ovarian cells, when transplanted under the kidney capsules, formed oocytes with a GFP-positive signal, which indicates that those cells were derived from skin-derived stem cells [37].

While oocytes differentiated from mice and human sources are well studied, there is limited data on bovine-derived oocytes. OLCs have been derived from bovine OSCs that express pluripotent germ cell markers (OCT4 and SOX2) and show alkaline phosphatase activity, enabling the identification of primordial germ cells [38]. Oocytes have been derived from follicular aspirates of female germline stem cells (faFGSCs) that are established from scarce cortical tissue without an STO feeder layer and co-cultured with granulosa cells to promote the growth and development of OLCs. Expression of specific markers was evaluated in this study using reverse transcription-polymerase chain reaction and immunofluorescence analyses. These results showed that human germinal vesicle GV oocytes could develop from faFGSCs in vitro under appropriate conditions [21].

Isolation of OSCs using morphology-based methods, such as colony formation, can be more effective than using FACS or MACS. Co-culturing OSCs with granulosa cells can help differentiate germline stem cells accurately during oocyte development [22]. Moreover, through in vitro reconstitution of the entire cycle of the mouse female germline, mature oocytes can be generated from ESCs and iPSCs. In this study, stem cells were differentiated to form oocytes under in vitro differentiation culture conditions, primary oocytes grow into germinal vesicle oocytes under in vitro growth culture conditions, and a robust number of metaphase II (MII) oocytes were produced under in vitro maturation culture conditions [14].

Differentiation of stem cells into ovarian follicles

Ovarian follicles are the main ovarian reserve for oocytes and have features of both germ and somatic cells. The oocytes mature into the MII stage in these unique cellular units [39] (Table 5). DAZL and BOULE promote the formation of meiotic germ cells, which can be induced to initiate folliculogenesis in vitro [40]. Human ESCs can differentiate into follicle-like cells (FLCs), which possess characteristics of both oocyte-like cells and granulosa cells. The structure of FLC is similar to that of the primary [40]. Even mouse ESCs can differentiate into FLCs that are similar to the primordial and primary follicles observed during meiosis in the ovary [41]. Further, under appropriate conditions, primordial follicles derived from human amniotic fluid stem cells produce estradiol and express genes that encoding steroidogenic enzymes. These primordial follicles strongly activate BMP15, which has a critical impact on oocyte maturation and fertilization, and express pluripotent markers (OCT4, NANOG, and ZP2) crucial for fertilization and preimplantation development [42].

Table 5.

Differentiation of stem cells into ovarian follicles

Culture	Markers	Origin	Species	Methods	Characters	References
Monolayer culture	SSEA, OCT4, SYCP1 ~ 3, REC8, STAG8, SMC1-β	mESC	Mouse	Immunofluorescence microscopy method	ESC-derived germ cells expressed SYCP3 but do not progress meiosis	[41]
	OCT4, SOX2, SSEA-4, TRA-1–60, CD117, NANOG, CD45, GDF9, BMP15, SCP3, DMC1, FOXL2, STELLA, FIGLA	hAFSC	Human amniotic fluid cells	RT-PCR	Female germ cells converted from hAFSCs grown in medium-containing cells	[42]
				Immunohistochemistry
	OCT4, NANOG, DAZL, PRDM14, AMH	hESC	Human	FACS	DAZL, BOULE regulate the exit from pluripotency and entry into meiosis	[40]
				Quantitative PCR DNA content analysis

Differentiation of stem cells into granulosa cells

Granulosa cells are critical for ovarian folliculogenesis. Granulosa cells can be derived from the differentiation of human amniotic epithelial cells, iPSCs, and ESCs (Table 6). Human amniotic epithelial cells restore ovarian function through the expression of specific markers [43]. Further, human iPSCs can differentiate into ovarian granulosa-like cells (OGLCs), which exhibits a fibroblast cell-like morphology and express ovarian granulosa cell markers. Therefore, iPSC-derived OGLCs are thought to reestablish ovarian function by promoting follicular maturation [25]. In another experiment, murine iPSCs were induced to form granulosa-like cells that synthesize and secrete estradiol in vitro. Radioimmunoassay and immunohistochemistry of murine iPSCs co-cultured with granulosa cells showed increased estradiol levels and follicle-stimulating hormone receptor (FSHR) expression in a time-dependent manner [26].

Table 6.

Differentiation of stem cells  into granulosa cells

Markers	Methods	Origin	Species	Characterizations	References
AMHR2, FOXL2, BRDU, CYP19A1, SOX9	FACS, RT-PCR, Microarray analysis, qRT-PCR	mESC human iPSC-derived EBs	FVB-mice	Identified and isolated GC-like cells from growth factor-free, gel-adherent EB cultures	[18]
BMP15, FMR1, INHA, FSHR, OCT4, NANOG, AMH, NOBOX, FOXO3, EIF2B, FIGLA, GDF9	RT-PCR, radioimmunoassay	iPSC ESC	4 week old female rats	Rat iPSCs and rat ESCs differentiated into GC-like cells during co-culture with GCs	[24]
SOX17, PDX1, PAX2, ODD1, SSEA-4, FOXL2, AMH, FSHR, AMHR2, LHR, OCT4, NANOG, CYP19A1	RT-PCR, Western blot analysis, Immunofluorescence staining, Flow cytometric analysis	hESC	Woman undergoing IVF	Differentiation of hESCs into intermediate plate mesoderm and finally into granulosa-like cells	[44]
FSHR	Immunocytochemistry, Radioimmunoassay, Flow cytometry assay	miPSCs	4 week old female C57BL/6 mice	By co-culturing with GCs, iPSCs were induced gradually to function as GC-like cells	[26]
FOXL2, WNT4, NR5AL, FST, KITL, FSHR, AMH, LHR, CYP19A1, STAR	FACS, RT-PCR	ESCs	Wild type C57BL/6 mice	Use of a Foxl2 gene promoter-driven fluorescent reporter system facilitates purification and study of granulosa cells at progressive stages of differentiation from ESC cultures	[17]
AKP, OCT4, SSEA-4, SOX2, NANOG, AMH, FSHR	Ovarian pathological analysis, H&E staining, ELISA, AKP assay, Immunofluorescent staining	iPSC	Human, Mice	iPSCs-derived OGLCs were capable of repairing ovarian injury and enhancing ovarian function	[25]
OCT4, NANOG, C-KIT, BLIMP1, STELLA, DAZL, VASA, STRA8, c-MOS, SCP1, SCP3	RT-qPCR, Immunohistochemical analysis	hAECs	C57BL/6 wild-type female mice	hAECs can differentiate into cells from all three embryonic tissue types	[43]

Additionally, OGLCs can be derived from rat ESCs and iPSCs by co-culturing these cells with granulosa cells in vitro through FSHR expression [24]. Remarkably, the differentiated granulosa cells showed premature ovarian insufficiency. In another experiment, human ESCs differentiated into granulosa-like cells that expressed FOXL2, CYP19A1, AMH, AMHR2, and FSHR. These human ESC-derived granulosa-like cells had a morphology similar to that of human luteinized granulosa cells. Therefore, human ESC-derived granulosa-like cells are similar to mature human ovarian granulosa cells [44].

Using the FOXL2 gene promoter-driven fluorescent reporter system, it was revealed that FOXL2 expressing cells are likely to be granulosa cells [17]. The FOXL2-positive cells also expressed FST, AMH, FSHR, WNT4, and KITL. Isolated and functional anti-Mullerian hormone receptor-positive granulosa-like cells were derived from murine EBs and evaluated for the development of sterogenic EBs using laser capture microscopy. These differentiated OGLCs expressed granulosa cell markers (CYP19A1, FOXL2, AMHR2, FSHR, and GJA1) and produced estradiol [18].

 Summary

This review discusses the overall oogenesis and folliculogenesis of the mammalian ovary. Although, there are opposing opinions regarding the existence of mammalian OSCs, we believe that they exist, based on the results of several studies on OSCs. Bone marrow, ovarian cortex, OSE, and skin-derived and pancreatic stem cells, are used as sources of OSCs. As OSCs are derived from various sources, the generation of OSCs remain uncertain. While most studies used FACS and MACS to isolate OSCs, they can also be isolated using differential adhesion-based methods.

However, it is uncertain how oocytes are generated from mammalian OSCs. Several cells are involved in the generation and development of OSCs. Most experiments on OSCs have been conducted using mice. It is now crucial to conduct experiments on non-human primates and humans and connect and interpret the data from these experimental systems [45–47].

In conclusion, our review shows research trends in oogenesis from OSCs and highlights the need for future research in this field. We believe that these findings can provide relevant insights for the treatment of female infertility.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical statement

There are no animal experiments carried out for this article.