The pluripotency of human embryonic stem cells (hESC) could have great potential for the development of cell replacement therapies. Previous studies have converged on the finding that OCT4, SOX2, and NANOG serve as key regulators in the maintenance of hESC. However, other signals that regulate hESC maintenance remain poorly studied. Here we describe a novel role of an RNA polymerase III (Pol III) subunit, POLR3G, in the maintenance of pluripotency in hESC. We demonstrate the presence of POLR3G in undifferentiated hESC, human induced pluripotent stem cells (hiPSC), and early mouse blastocysts. Downregulation of POLR3G is observed on differentiation of hESC and hiPSC, suggesting that POLR3G can be used as a molecular marker to readily identify undifferentiated pluripotent stem cells from their differentiated derivatives. Using an inducible shRNA lentiviral system, we found evidence that decreased levels of POLR3G result in loss of pluripotency and promote differentiation of hESC to all three germ layers but have no effect on cell apoptosis. On the other hand, overexpression of POLR3G has no effect on pluripotency and apoptosis in undifferentiated hESC. Interestingly, hESC expressing elevated levels of POLR3G are more resistant to differentiation. Furthermore, our experimental results show that POLR3G is a downstream target of OCT4 and NANOG, and our pharmacological study indicated that POLR3G expression can be readily regulated by the Erk1/2 signaling pathway. This study is the first to show an important role of POLR3G in the maintenance of hESC, suggesting a potential role of Pol III transcription in regulating hESC pluripotency. STEM CELLS

BackgroundHuman embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many of the downstream targets of these transcription factors are not well characterized. Furthermore, it remains unknown whether additional novel stem cell factors are involved in the establishment and maintenance of the stem cell state.Methodology/Principal FindingsHere we show that a novel gene, L1TD1 (also known as FLJ10884 or ECAT11), is abundantly expressed in undifferentiated hESC. Differentiation of hESC via embryoid body (EB) formation or BMP4 treatment results in the rapid down-regulation of L1TD1 expression. Furthermore, populations of undifferentiated and differentiated hESC were sorted using the stem cell markers SSEA4 and TRA160. Our results show that L1TD1 is enriched in the SSEA4-positive or TRA160-positive population of hESC. Using chromatin immunoprecipitation we found enriched association of Nanog to the predicted promoter region of L1TD1. Furthermore, siRNA-mediated knockdown of Nanog in hESC also resulted in downregulation of L1TD1 expression. Finally, using luciferase reporter assay we demonstrated that Nanog can activate the L1TD1 upstream promoter region. Altogether, these results provide evidence that L1TD1 is a downstream target of Nanog.Conclusion/SignificanceTaken together, our results suggest that L1TD1 is a downstream target of Nanog and represents a useful marker for identifying undifferentiated hESC.

Valosin Containing Protein (VCP) disease is an autosomal dominant disorder caused by mutations in the VCP gene and is associated with progressive muscle weakness and atrophy. Affected individuals exhibit striking scapular winging due to shoulder girdle weakness. Currently, there are no treatments available and patients are dying early from cardiac and respiratory failure, typically in their 40's and 50's. The generation of disease-specific induced pluripotent stem cells (iPSC) offers a novel platform to investigate mechanisms of VCP disease and potential treatments similar to other disease models including Amyotrophic Lateral Sclerosis (ALS), Duchenne muscular dystrophy (DMD), Parkinson's disease (PD), Alzheimer's disease (AD), Best Disease (BD), and type I juvenile diabetes mellitus (T1DM). Herein, we report the generation and characterization of a human iPSC line to examine the cellular and molecular processes underlying VCP disease. The VCP iPSC line expressed specific pluripotency markers NANOG, SSEA4, OCT-4, TRA-1-81 and exhibited characteristic morphology. We differentiated the human iPSC cell line into a neuronal lineage confirmed by TUJ-1 staining, a neuronal class III β-tubulin marker. We detected higher protein expression levels of ubiquitin (Ub), TAR DNA-binding protein-43 (TDP-43), Light Chain 3-I/II (LC3), p62/SQSTM1, and optineurin (OPN) in the iPSC neural lineage compared to the control neural line. Collectively, our results demonstrate that patient-specific iPSC technology may provide useful disease modeling for understanding the complex mechanisms and for developing novel treatments of VCP and related disorders.