Oct4 Expression In Cells Research Articles

Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer

Nuclear transfer (NT) is associated with epigenetic reprogramming of donor cells. Expression of certain genes in these cells might facilitate their expression in the NT embryo. This research was aimed to investigate the effect of constitutive expression of OCT4 in bovine somatic cells used for NT on the developmental potential of derived cloned embryos as well as in the expression of pluripotency markers in the Day-7 resulting embryos. Cloned blastocysts were generated from five cell lines that expressed OCT4. Pools of blastocysts were screened to detect OCT4, SOX2, and NANOG by qPCR. In vitro-fertilized time-matched blastocysts were used as controls. The development potential was assessed on the basis of blastocysts rate; grading and total cell counts at Day 7. OCT4 expression in the cell lines positively correlates with blastocysts rate (r = 0.92; p = 0.02), number of grade I blastocysts (r = 0.96; p = 0.01), and total cell number (r = 0.98; p = 0.002). The high expression of OCT4 in the cell line did not improve the final outcome of cloning. Somatic expression of OCT4 lead to increased expression of OCT4 and SOX2 in cloned grade I blastocysts; however, there was a bigger variability in OCT4 and SOX2 (p = 0.03; p = 0.02) expression in the embryos generated from cells expressing highest levels of OCT4. Probably the higher variability in OCT4 expression in cloned embryos is due to incorrect reprogramming and incapability of the oocyte to correct for higher OCT4 levels. For that reason, we concluded that OCT4 expression in somatic cells is not a good prognosis marker for selecting cell lines.

Brief Report: Analysis of Endogenous Oct4 Activation during Induced Pluripotent Stem Cell Reprogramming Using an Inducible Oct4 Lineage Label

The activation of endogenous Oct4 transcription is a key step in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells but until now it has been difficult to analyze this critical event in the reprogramming process. We have generated a transgenic mouse that expresses the tamoxifen-inducible Cre recombinase MerCreMer under the control of the endogenous Oct4 locus, enabling lineage tracing of Oct4 expression in cells in vivo or in vitro, during either reprogramming or differentiation. Using this novel resource, we have determined the timing and outcome of endogenous Oct4 induction during fibroblast reprogramming. We show that both the initiation of this key reprogramming step and the ability of cells activating endogenous Oct4 expression to complete reprogramming are not influenced by the presence of exogenous c-Myc, although the overall efficiency of the process is increased by c-Myc. Oct4 lineage tracing reveals that new reprogramming events continue to initiate over a period of 3 weeks. Furthermore, the analysis of mixed colonies, where only a subset of daughter cells induce endogenous Oct4 expression, indicates the role of unknown, stochastic events in the progression of reprogramming from the initial events to a pluripotent state. Our transgenic mouse model and cells derived from it provide powerful and precise new tools for the study of iPS cell reprogramming mechanisms and have wider implications for the investigation of the role of Oct4 during development.

Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4+ very small embryonic-like stem cells

Recently, we identified in adult tissues a population of Oct4+SSEA-1+Sca-1+lin-CD45- very small embryonic-like stem cells (VSELs). First, to address recent controversies on Oct4 expression in cells isolated from adult organs we show here an evidence that Oct4 promoter in bone marrow (BM)-derived VSELs has an open chromatin structure and is actively transcribed. Next, to explain VSELs quiescence and lack of teratoma formation we demonstrate a unique DNA methylation pattern at some developmentally crucial, imprinted-genes, showing hypomethylation/erasure of imprints in paternally methylated and hypermethylation of imprints in maternally methylated ones. These epigenetic characteristics leading to upregulation in VSELs of H19 and p57KIP2 (also known as Cdkn1c) and repression of Igf2 and Rasgrf1 explain VSEL's quiescent status. Interestingly, this unique pattern in imprinted-genes methylation is reverted in co-cultures with a C2C12 supportive cell-line when VSELs are induced to form VSEL-derived spheres (VSEL-DSs) enriched for stem cells able to differentiate into all three germ layers. Therefore, we suggest that the proliferative/developmental potential of Oct4+ VSELs is epigenetically regulated by expression of Oct4 and some imprinted-genes, and postulate that restoring the proper methylation pattern of imprinted-genes will be crucial step for employing these cells in regenerative medicine.