14 research outputs found
Overexpression of Nuclear Receptor 5A1 Induces and Maintains an Intermediate State of Conversion between Primed and Naive Pluripotency
Naive and primed human pluripotent stem cells (hPSCs) have provided useful insights into the regulation of pluripotency. However, the molecular mechanisms regulating naive conversion remain elusive. Here, we report intermediate naive conversion induced by overexpressing nuclear receptor 5A1 (NR5A1) in hPSCs. The cells displayed some naive features, such as clonogenicity, glycogen synthase kinase 3β, and mitogen-activated protein kinase (MAPK) independence, expression of naive-associated genes, and two activated X chromosomes, but lacked others, such as KLF17 expression, transforming growth factor β independence, and imprinted gene demethylation. Notably, NR5A1 negated MAPK activation by fibroblast growth factor 2, leading to cell-autonomous self-renewal independent of MAPK inhibition. These phenotypes may be associated with naive conversion, and were regulated by a DPPA2/4-dependent pathway that activates the selective expression of naive-associated genes. This study increases our understanding of the mechanisms regulating the conversion from primed to naive pluripotency
In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells
BACKGROUND: Mouse 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 FINDINGS: To 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
The effect of BMP4, RA and SCF on germ cell differentiation in monkey EBs.
<p>(A) <i>VASA</i> expression in EBs cultured in the presence or absence of BMP4 (100 ng/ml), RA (1 µM), and SCF (100 ng/ml) was compared using quantitative RT-PCR. The expression levels of <i>VASA</i> in treated EBs are shown relative to <i>VASA</i> expression in ES cells (day 0). The data represent the means±standard deviation from triplicate PCR assays. (B) The expression of <i>SCP1</i> and <i>SCP3</i> in monkey testis (5 years old), ES cells (ES), and developing EBs (days 14, 21, and 28) in the presence or absence of BMP4 (100 ng/ml), RA (1 µM), and SCF (100 ng/ml) was examined using RT-PCR. <i>GAPDH</i> was used as an internal control. Statistical significance was tested by Student's <i>t</i>-test. Asterisk, <i>P</i><0.01.</p
The expression of germ cell marker genes in tissues of the cynomolgus monkey.
<p>The expression of germ cell marker genes in monkey ES cells and tissues was examined using an RT-PCR analysis. <i>GAPDH</i> was used as an internal control.</p
Morphological features of cynomolgus monkey testes, and expression of VASA protein.
<p>(A–C) Sections of testes of 3-year-old (A) and 5-year-old monkeys (B,C) were stained with hematoxylin and eosin. Sg, spermatogonium; P, pachytene spermatocyte; Sd, spermatid. (D–Q) Expression of VASA, DAZL, and SCP1 proteins during spermatogenesis in cynomolgus monkeys. Sections of 3-year-old (D,E) and 5-year old testes (F–Q) were examined by immunostaining. The nuclei were stained with Hoechst 33258. Merged images were also shown (E; VASA, red; Hoechst, white), (H, I; VASA, red; DAZL, green; Hoechst, white), (L,M; VASA, red; SCP1, green; Hoechst, white), (P,Q; DAZL, red; SCP1, green; Hoechst, white). VASA expression was observed in spermatogonia in the 3-year-old testis (E, red), and in spermatogonia (open arrowhead), spermatocytes (arrow), and early spermatids (arrowhead) in the 5-year-old testis (I, M, red). The expression of VASA, DAZL, and SCP1 proteins was all detected in spermatocytes (arrow; I, M, Q). The dotted lines indicate the basement membranes of the seminiferous tubules. The scale bar is 25 µm.</p
The expression of germ cell marker genes in ES cells of cynomolgus monkey during EB formation.
<p>(A) The expression patterns of germ cell marker genes in monkey testis (5 years old), mouse embryonic fibroblast (MEF), monkey ES cells (ES), and developing EBs (days 3, 7, 14, 21, and 28) were examined using an RT-PCR analysis. <i>GAPDH</i> was used as an internal control. (B–N) An immunocytochemical analysis of ES cells and day 14 EBs. ES cells were doubly immunostained with anti-OCT-4 and anti-VASA antibodies (B). Left column, OCT-4; middle column, VASA; right column, OCT-4 (green) and VASA (red) merged with Hoechst 33258 (white) staining. The sections of day 14 EBs were doubly immunostained with anti-SSEA1 and anti-OCT-4 (C–H) antibodies, or anti-OCT-4 and anti-VASA (I–N) antibodies. The expression of each protein is shown in left and middle columns. Merged images with Hoechst 33258 (white) staining are also shown in right column (E, H; OCT-4, red; SSEA1, green; K, N; OCT-4, green; VASA, red). The scale bars are 100 µm (B) and 25 µm (C–N), respectively.</p
The effect of mouse testicular and ovarian cell-conditioned media on germ cell differentiation in monkey EBs.
<p>(A) The <i>VASA</i> expression in ES cells (day 0) and EBs (days 7, 14, 21, and 28) in non-conditioned medium, testicular, or ovarian cell-conditioned medium was compared using quantitative RT-PCR. The expression levels of <i>VASA</i> in treated EBs are shown relative to <i>VASA</i> expression in ES cells (day 0). The data represent the means±standard deviation from triplicate PCR assays. (B) Expression of <i>SCP1</i> and <i>SCP3</i> in monkey testis (5 years old), ES cells (ES), and developing EBs (days 14, 21, and 28) in non-conditioned medium, testicular, or ovarian cell-conditioned medium was examined using RT-PCR. <i>GAPDH</i> was used as an internal control. Statistical significance was tested by Student's <i>t</i>-test. Asterisk, <i>P</i><0.01.</p