OCT4 Silencing Triggers Its Epigenetic Repression and Impairs the Osteogenic and Adipogenic Differentiation of Mesenchymal Stromal Cells.

Mechanisms mediating mesenchymal stromal/stem cells' (MSCs) multipotency are unclear. Although the expression of the pluripotency factor OCT4 has been detected in MSCs, whether it has a functional role in adult stem cells is still controversial. We hypothesized that a physiological expression level of OCT4 is important to regulate MSCs' multipotency and trigger differentiation in response to environmental signals. Here, we specifically suppressed OCT4 in MSCs by using siRNA technology before directed differentiation. OCT4 expression levels were reduced by 82% in siOCT4-MSCs, compared with controls. Interestingly, siOCT4-MSCs also presented a hypermethylated OCT4 promoter. OCT4 silencing significantly impaired the ability of MSCs to differentiate into osteoblasts. Histologic and macroscopic analysis showed a lower degree of mineralization in siOCT4-MSCs than in controls. Moreover, OCT4 silencing prevented the up-regulation of osteoblast lineage-associated genes during differentiation. Similarly, OCT4 silencing resulted in decreased MSC differentiation potential towards the adipogenic lineage. The accumulation of lipids was reduced 3.0-fold in siOCT4-MSCs, compared with controls. The up-regulation of genes engaged in the early stages of adipogenesis was also suppressed in siOCT4-MSCs. Our findings provide evidence of a functional role for OCT4 in MSCs and indicate that a basal expression of this transcription factor is essential for their multipotent capacity.

Exogenous human OKSM factors maintain pluripotency gene expression of bovine and porcine iPS-like cells obtained with STEMCCA delivery system.

OBJECTIVES: The use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated. RESULTS: Protocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals' iPS cells.

Characterization of Oct4-GFP transgenic mice as a model to study the effect of environmental estrogens on the maturation of male germ cells by using flow cytometry.

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17α-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development.

Participation of OCT3/4 and beta-catenin during dysgenetic gonadal malignant transformation.

Gonadoblastoma (GB) is an in situ tumor consisting of a heterogeneous population of mature and immature germ cells, other cells resembling immature Sertoli/granulosa cells, and Leydig/lutein-like cells, may also be present. GB almost exclusively affects a subset of patients with intersex disorders and in 30% of them overgrowth of the germinal component of the tumor is observed and the lesion is term dysgerminoma/seminoma. Several pathways have been proposed to explain the malignant process, and abnormal OCT3/4 expression is the most robust risk factor for malignant transformation. Some authors have suggested that OCT3/4 and beta-catenin might both be involved in the same oncogenic pathway, as both genes are master regulators of cell differentiation and, overexpression of either gene may result in cancer development. The mechanism by which beta-catenin participates in GB transformation is not completely clear and exploration of the E-cadherin pathway did not conclusively show that this pathway participated in the molecular pathogenesis of GB. Here we analyze seven patients with mixed gonadal dysgenesis and GB, in an effort to elucidate the participation of beta-catenin and E-cadherin, as well as OCT3/4, in the oncogenic pathways involved in the transformation of GB into seminoma/dysgerminoma. We conclude that the proliferation of immature germ cells in GB may be due to an interaction between OCT3/4 and accumulated beta-catenin in the nuclei of the immature germ cells.