Induced Pluripotent Stem Cells from Ovarian Tissue.

Yamanaka and colleagues revolutionized stem cell biology and regenerative medicine by observing that somatic cells can be reprogrammed into pluripotent stem cells. Evidence indicates that induced pluripotent stem (iPS) cells retain epigenetic memories that bias their spontaneous differentiation into the originating somatic cell type, therefore epigenetic memory may be exploited to improve tissue specific regeneration. We recently showed that iPS cells reprogrammed from ovarian granulosa cells using mouse and human tissue overwhelmingly differentiate homotypically into ovarian steroidogenic and primordial germ cells. Herein we detail a protocol for the culture of human ovarian granulosa cells. We review approaches for reprogramming human ovarian granulosa cells into iPS cells. Standard methods to induce pluripotency are outlined, concentrating on integrative retroviruses. Additionally, alternative protocols for lentivirus and Sendai virus are provided. Each approach has inherent limitations, such as reprogramming efficiency, insertional mutagenesis, and partial reprogramming. Major advances continue to be made in somatic cell reprogramming to identify an optimal approach and utilization in cell-based therapies. © 2017 by John Wiley & Sons, Inc.

The transcription factor, Nuclear factor, erythroid 2 (Nfe2), is a regulator of the oxidative stress response during Danio rerio development.

Development is a complex and well-defined process characterized by rapid cell proliferation and apoptosis. At this stage in life, a developmentally young organism is more sensitive to toxicants as compared to an adult. In response to pro-oxidant exposure, members of the Cap'n'Collar (CNC) basic leucine zipper (b-ZIP) transcription factor family (including Nfe2 and Nfe2-related factors, Nrfs) activate the expression of genes whose protein products contribute to reduced toxicity. Here, we studied the role of the CNC protein, Nfe2, in the developmental response to pro-oxidant exposure in the zebrafish (Danio rerio). Following acute waterborne exposures to diquat or tert-buytlhydroperoxide (tBOOH) at one of three developmental stages, wildtype (WT) and nfe2 knockout (KO) embryos and larvae were morphologically scored and their transcriptomes sequenced. Early in development, KO animals suffered from hypochromia that was made more severe through exposure to pro-oxidants; this phenotype in the KO may be linked to decreased expression of alas2, a gene involved in heme synthesis. WT and KO eleutheroembryos and larvae were phenotypically equally affected by exposure to pro-oxidants, where tBOOH caused more pronounced phenotypes as compared to diquat. Comparing diquat and tBOOH exposed embryos relative to the WT untreated control, a greater number of genes were up-regulated in the tBOOH condition as compared to diquat (tBOOH: 304 vs diquat: 148), including those commonly found to be differentially regulated in the vertebrate oxidative stress response (OSR) (e.g. hsp70.2, txn1, and gsr). When comparing WT and KO across all treatments and times, there were 1170 genes that were differentially expressed, of which 33 are known targets of the Nrf proteins Nrf1 and Nrf2. More specifically, in animals exposed to pro-oxidants a total of 968 genes were differentially expressed between WT and KO across developmental time, representing pathways involved in coagulation, embryonic organ development, body fluid level regulation, erythrocyte differentiation, and oxidation-reduction, amongst others. The greatest number of genes that changed in expression between WT and KO occurred in animals exposed to diquat at 2h post fertilization (hpf). Across time and treatment, there were six genes (dhx40, cfap70, dnajb9b, slc35f4, spi-c, and gpr19) that were significantly up-regulated in KO compared to WT and four genes (fhad1, cyp4v7, nlrp12, and slc16a6a) that were significantly down-regulated. None of these genes have been previously identified as targets of Nfe2 or the Nrf family. These results demonstrate that the zebrafish Nfe2 may be a regulator of both primitive erythropoiesis and the OSR during development.