Isolation of Mammalian Oogonial Stem Cells by Antibody-Based Fluorescence-Activated Cell Sorting

Abstract

The ability to isolate and subsequently culture mitotically active female germ cells from adult ovaries, referred to as either oogonial stem cells (OSCs) or adult female germline stem cells (aFGSCs), has provided a robust system to study female germ cell development under multiple experimental conditions, and in many species. Flow cytometry or fluorescence-activated cell sorting (FACS) is an integral part of many isolation and characterization protocols. Here, we provide methodological details for antibody-based flow cytometric isolation of OSCs using antibodies specific for external epitopes of the proteins Ddx4 or Ifitm3, alone or in combination with the use of fluorescent reporter mice. Beginning with sample preparation, we provide point-by-point instructions to guide researchers on how to isolate OSCs using flow cytometry.

Genetic studies in mice directly link oocytes produced during adulthood to ovarian function and natural fertility

“…As important as these observations are, the current lack of a gene promoter that is exclusively expressed in OSCs, but not in differentiating premeiotic germ cells or oocytes, does not enable unequivocal identification of OSCs as the cells responsible for supporting postnatal oogenesis. However, the independent verification of the presence of OSCs in the ovaries of a growing number of mammalian species 10 , 12 – 35 , 46 , 78 , the ability of OSCs to generate new oocytes and functionally competent eggs in adult females 12 , 15 , 16 , 20 , 21 , 25 , 32 , 34 , 35 (Supplementary Fig. S1 ), and experimental evidence that resident multi-potent stem cells in adult ovaries are unable to generate oocytes 79 , collectively support that OSCs are the most logical and likely source of new oocytes formed during adulthood.…”

“…For most experiments (except as indicated below), OSCs were isolated from ovaries of young adult mice (2–3 months of age) by FACS using a C-terminal DDX4-specific antibody (ab13840, Abcam). The cells were analyzed immediately or established in culture without somatic feeder cells, as described 16 , 17 , 46 , 93 . Purified mouse OSCs propagated under these conditions spontaneously differentiate into IVD-oocytes for up to 72 h after passage until confluence is regained, and the number of IVD-oocytes generated by a fixed number of OSCs seeded per well remains relatively constant over successive passages 16 , 17 , 19 .…”

“…Expression of Ifitm3 is used frequently as an endpoint in studies of primitive germ cells, and Ifitm3 antibody-based sorting of embryonic primordial germ cells is well documented 44 , 45 . Expression of Ifitm3 at both the mRNA and protein levels can be detected in OSCs of multiple species 12 , 16 , 21 , 30 , and antibodies against an extracellular domain of Ifitm3 have been used to sort OSCs from mouse 14 , 46 and rat 21 ovaries. These cells, like those sorted using Ddx4 antibodies, generate functional eggs and offspring following transplantation 21 .…”

Implications and Current Limitations of Oogenesis from Female Germline or Oogonial Stem Cells in Adult Mammalian Ovaries

“…We will begin with the method employed to obtain OSCs from ovarian cortical tissue of both pre- and post-menopausal women [73,97,98,99,101]. The underlying approach relies on the use of an antibody against the C-terminus of the germ cell protein, DEAD-box polypeptide 4 (DDX4) [109,110,111], to identify and purify those cells with externalized (extracellular) exposure of this specific domain of the protein by fluorescence-activated cell sorting (FACS) or magnetic-assisted cell sorting (MACS) [53,68,73,112]. The cells obtained from human ovarian tissue by this method exhibit many characteristic germline features that closely align with OSCs isolated from adult mouse ovaries.…”

Age-related changes in gene expression patterns of immature and aged rat primordial follicles

“…Other than the fixed number of oocytes in the adult ovaries, the existence of female germ line or oogonical stem cells (OSC) in the adult ovaries of mice, rats, and human have been reported [Grieve et al 2015;White et al 2012]. Although the conflicting results on isolation and confirmation of OSCs [Navaroli et al 2016;Zarate-Garcia et al 2016] and contrary observations on the source of these cells (native to ovary or migrated from other tissues) [Dunlop et al 2013;Johnson et al 2005], currently, there is no data available on the possible role of OSCs in normal physiological function of ovary including fertility. It was argued that even if OSCs can support regeneration of oocytes, his may be restricted to some extent, because the age-related decrease in follicle number is not disputed and adult neo-oogenesis may be considered insignificant due to the large number of fixed oocytes [Zarate-Garcia et al 2016].…”