Kerr CL, Hill C

Kerr CL, Hill C.M, Blumenthal P, Gearhart J.D. in (Urochordata), BS-PL10 modulates this animal’s blastogenic cycle, increasing from blastogenic stage A to blastogenic stage D [28] while a sharp decrease in BS-PL10 expression occurs during organogenesis such that the highest levels of expression is observed in multipotent soma and germ cells. Also, Ddx3x heterozygous female mice exhibits placental abnormalities during development and is embryonic lethal [29]. In addition, loss of ddx3x results in widespread apoptosis due to enhanced GW 542573X DNA damage and cell cycle arrest [29]. Thus, together GW 542573X with the evolutionary conservation of DDX3 [30], evidence points to this as an ancestral gene with defined functional roles both in self-renewal and pluripotency. Here, we report that DDX3 promotes stem cell maintenance. Specifically, we show that undifferentiated embryonic stem cells (ESC) and embryonal carcinoma cells (ECCs) express high levels of DDX3 compared to differentiated cells. Notably, when DDX3 activities were Rabbit polyclonal to Osteopontin perturbed, we observed a drastic decrease in the proliferation of undifferentiated stem cells along with an increase in cellular differentiation. Moreover, we also confirmed that inhibiting DDX3 activity prevents teratoma formation in NOD-scidIL-2Rnull (NOG) mice. Taken together, our results indicate that DDX3 is an integral component of stem cell character and regulating DDX3 activity could be used to control differentiation and pluripotency. RESULTS DDX3 expression decreases with differentiation in human ESCs and ECCs Following gene expression analysis of pluripotent ESCs and unipotent progenitors of embryonic germ cells (EGCs) and ECCs known as primordial germ cells (PGCs), DDX3 was identified as one of a few genes that showed differential expression between these two cell types. To confirm this finding, qRT-PCR analysis was performed, which showed that DDX3 mRNA expression is significantly higher in ESCs and ECCs than in their differentiated counterparts of neural lineage (NRN) and human fetal fibroblasts (hFF) compared to primordial germ cells (baseline), which are the unipotent, or more differentiated progenitors of EGCs and ECCs (Figure ?(Figure1).1). This was further corroborated by using three independent DDX3 specific primer sets (data not shown). Importantly, evidence comparing EGC to the PGC from which they are derived indicates that DDX3 may be involved in the initial stages driving pluripotency. Open in a separate window Figure 1 Expression of DDX3 in pluripotent and differentiated cell linesDDX3 expression is lower in differentiated cells (FF: human fetal fibroblasts; ECC Neuro: Neural differentiated hECCs) and higher in pluripotent stem cells (hEGCs, hECCs and hESCs). Relative expression of was compared to -actin as the endogenous control. Ct method was also employed using the unipotent germ cell progenitor cells, PGCs as the baseline value (= 3, 0.05). Altered DDX3 expression levels following differentiation of ESCs and ECCs As DDX3 levels were altered following differentiation, we analyzed DDX3 expression by immunofluorescence to determine the expression pattern at the cellular level. As show in Figure ?Figure2,2, DDX3 expression was significantly reduced after differentiation of ECCs demonstrating that undifferentiated ECCs that express OCT4 (Figure ?(Figure2A)2A) also express DDX3 (Figure ?(Figure2B).2B). More importantly, when cultured under neural-inducing conditions DDX3 expression is ablated (Figure ?(Figure2E).2E). This is evident by the lack of DDX3 expression in cells (Figure ?(Figure2E)2E) which have little or no expression of the pluripotent cell surface marker TRA-1-60 (Figure ?(Figure2D)2D) compared to the undifferentiated ECCs known GW 542573X to express both TRA-1-60 and OCT4. These results indicate GW 542573X that DDX3 expression is concomitant with.