OCT-4 transcription factors play an important role in maintaining the pluripotent state of embryonic stem cells and may prevent expression of genes activated during differentiation. Human OCT-4 isoform mRNAs encode proteins that have identical POU DNA binding domains and C-terminal domains but differ in their N-terminal domains. We report here the cloning and characterization of the human OCT-4B isoform. Human OCT-4B cDNA encodes a 265-amino acid protein with a predicted molecular mass of 30 kDa. Embryonic stem (ES) cellbased complementation assays using ZHBTc4 ES cells showed that unlike human OCT-4A, OCT-4B cannot sustain ES cell self-renewal. In addition, OCT-4B does not bind to a probe carrying the OCT-4 consensus binding sequence, and we demonstrate that two separate regions of its N-terminal domain are responsible for inhibiting DNA binding. We also demonstrate that OCT-4B is mainly localized to the cytoplasm. Overexpression of OCT-4B did not activate transcription from OCT-4-dependent promoters, although OCT-4A did as reported previously. Furthermore, transcriptional activation by human OCT-4A was not inhibited by co-expression of OCT-4B. Taken together, these data suggest that the DNA binding, transactivation, and abilities to confer self-renewal of the human OCT-4 isoforms differ.

SummarySchwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system.

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Costello syndrome is linked to activating mutations of a residue in the p-loop or the NKCD/SAK motifs of Harvey Ras (HRas). More than 10 HRas mutants that induce Costello syndrome have been identified; G12S HRas is the most prevalent of these. However, certain HRas p-loop mutations also are linked to cancer formation that are exemplified with G12V HRas. Despite these relations, specific links between types of HRas mutations and diseases evade definition because some Costello syndrome HRas p-loop mutations, such as G12S HRas, also often cause cancer. This study established novel kinetic parameter-based equations that estimate the value of the cellular fractions of the GTP-bound active form of HRas mutant proteins. Such calculations differentiate between two basic kinetic mechanisms that populate the GTP-bound form of Ras in cells. (i) The increase in GTP-bound Ras by the HRas mutation-mediated perturbation of the intrinsic kinetic characteristics of Ras. This generates a broad spectrum of the population of the GTP-bound form of HRas that typically causes Costello syndrome. The upper end of this spectrum of HRas mutants, as exemplified by G12S HRas, can also cause cancer. (ii) The increase in GTP-bound Ras because the HRas mutations perturb the p120GAP action on Ras. This causes production of a significantly high population of the only GTP-bound form of HRas linked merely to cancer formation. The HRas mutant G12V belongs to this category.

Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the ‘stemness’ of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation.

The Oct-4 gene encodes a transcription factor that is expressed in embryonic stem (ES) cells and germ cells. Oct-4 is known to function as a transcriptional activator of genes involved in maintaining an undifferentiated totipotent state and possibly in preventing expression of genes activated during differentiation. In addition, it is a putative proto-oncogene and a critical player in the genesis of human testicular germ cell tumors. Although much effort has gone toward characterizing Oct-4, there is still little known about the molecular mechanisms and the proteins that regulate Oct-4 function. To identify cofactors that control Oct-4 function in vivo, we used a recently developed bacterial two-hybrid screening system and isolated a novel ES cell-derived cDNA encoding Ewing's sarcoma protein (EWS). EWS is a proto-oncogene and putative RNA-binding protein involved in human cancers. By using glutathione-S-transferase (GST) pull-down assays, we were able to confirm the interaction between Oct-4 and EWS in vitro, and moreover, coimmunoprecipitation and colocalization studies have shown that these proteins also associate in vivo. We have mapped the EWS-interacting region to the POU domain of Oct-4. In addition, three independent sites on EWS are involved in binding to Oct-4. In this study, we report that Oct-4 and EWS are coexpressed in the pluripotent mouse and human ES cells. Consistent with its ability to bind to and colocalize with Oct-4, ectopic expression of EWS enhances the transactivation ability of Oct-4. Moreover, a chimeric protein generated by fusion of EWS (1-295) to the GAL4 DNA-binding domain significantly increases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of a strong activation domain within EWS. Taken together, our results suggest that Oct-4-mediated transactivation is stimulated by EWS. Stem Cells 2005;23:738-751

The t(6;22)(p21;q12) translocation associated with human bone and soft-tissue tumours results in a chimaeric molecule fusing the NTD (N-terminal domain) of the EWS (Ewing's sarcoma) gene to the CTD (C-terminal domain) of the Oct-4 (octamer-4) embryonic gene. Since the N-terminal domains of EWS and Oct-4 are structurally different, in the present study we have assessed the functional consequences of the EWS-Oct-4 fusion. We find that this chimaeric gene encodes a nuclear protein which binds DNA with the same sequence specificity as the parental Oct-4 protein. Comparison of the transactivation properties of EWS-Oct-4 and Oct-4 indicates that the former has higher transactivation activity for a known target reporter gene containing Oct-4 binding. Deletion analysis of the functional domains of EWS-Oct-4 indicates that the EWS (NTD), the POU domain and the CTD of EWS-Oct-4 are necessary for full transactivation potential. EWS-Oct-4 induced the expression of fgf-4 (fibroblast growth factor 4) and nanog, which are potent mitogens as well as Oct-4 downstream target genes whose promoters contain potential Oct-4-binding sites. Finally, ectopic expression of EWS-Oct-4 in Oct-4-null ZHBTc4 ES (embryonic stem) cells resulted in increased tumorigenic growth potential in nude mice. These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS-Oct-4 chimaeric protein and that fusion of the EWS NTD to the Oct-4 DNA-binding domain produces a transforming chimaeric product.

Many elderly people suffer from hematological diseases known to be highly age-dependent. Hematopoietic stem cells (HSCs) maintain the immune system by producing all blood cells throughout the lifetime of an organism. Recent reports have suggested that HSCs are susceptible to age-related stress and gradually lose their self-renewal and regeneration capacity with aging. HSC aging is driven by cell-intrinsic and -extrinsic factors that result in the disruption of the immune system. Thus, the study of HSC aging is important to our understanding of age-related immune diseases and can also provide potential strategies to improve quality of life in the elderly. In this review, we delineate our understanding of the phenotypes, causes, and molecular mechanisms involved in HSC aging.