Abstract
BackgroundMouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro.Methods and FindingsTo explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene.Conclusion VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.
Highlights
Embryonic stem (ES) cells are pluripotent and can be used to study germ cell development [1,2,3,4,5]
VASA is a valuable gene for the detection of germ cells differentiated from embryonic stem (ES) cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo
Expression of germ cell markers in the tissues from the cynomolgus monkey To examine the expression patterns of germ cell markers in tissues of the cynomolgus monkey, we first performed RT-PCR analysis for germ cell marker genes reported in mice and humans, including PR domain containing 1, with ZNF domain (PRDM1, known as BLIMP1) [16], PR domain containing 14 (PRDM14) [17], protein arginine methyltransferase 5 (PRMT5) [18], DPPA3 [19,20,21], IFITM3 [20,22], growth and differentiation factor 3 (GDF3) [23,24], c-KIT [25,26], chemokine (C-X-C motif) receptor 4 (CXCR4) [27,28], NANOS1-3 [29,30,31], deleted in azoospermia-like (DAZL) [3235], VASA, PIWI family genes (PIWIL1 and PIWIL2, known as HIWI and HILI in humans, respectively) [36,37,38], Mut-L Homologue-1 (MLH1) [39,40], synaptonemal complex protein 1 (SCP1) [41,42,43,44], and SCP3 [42,45,46] (Figure 1)
Summary
Embryonic stem (ES) cells are pluripotent and can be used to study germ cell development [1,2,3,4,5]. Recent studies have demonstrated that mouse ES cells can differentiate into female and male germ cells in vitro, producing ovarian follicle-like structures [1,4] and testicular germ cells [2,3]. Stella-related (STELLAR), growth and differentiation factor 3 (GDF3), NANOS, cKIT, and DAZL, have been examined as well [5,12] These germ cell marker genes are not appropriate for detecting germ cell differentiation from mouse and human ES cells because these genes are expressed in both ES cells and germ cells. In order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro
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