Abstract
BackgroundWe have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes.Methodology/Principal FindingsWhen induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs.SignificanceThe ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.
Highlights
The process of early gametogenesis requires a succession of coordinated steps, including primordial germ cell (PGC) specification, migration to and colonization of the gonadal ridges, proliferation, and differentiation into more mature gametes [1]
A subpopulation of the round cells gradually detached and grew as a separate non-adherent population of single and clustered cells. This morphology is consistent with the typically oval or round shape, irregular or ‘‘blebbed’’ contour, and large nucleus associated with porcine PGCs dissociated from gonadal ridges [38] or murine PGCs [39], which are known to be larger in size compared to somatic cells
While it has been shown that cells resembling PGCs can be differentiated from embryonic stem cells (ESCs) in vitro, to our knowledge, the current investigation is the first to characterize PGC-like cells generated from the induced differentiation of somatic-derived stem cells
Summary
The process of early gametogenesis requires a succession of coordinated steps, including primordial germ cell (PGC) specification, migration to and colonization of the gonadal ridges, proliferation, and differentiation into more mature gametes [1]. Only a few studies conducted with ESCs [19,20,23] have focused on characterizing the germ cell precursors that arise in these cultures, and a direct link between PGC-like cells and later-stage germ cells in vitro has yet to be conclusively established. Primordial germ cells (PGCs), which must be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes
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