Epigenetic regulation of gene expression in porcine epiblast,hypoblast, trophectoderm and epiblast-derived neural progenitor cells

After fertilization, lineage specification is governed by a complicated molecular network in which permissiveness and repression of expression of pluripotency- and differentiation-associated genes are regulated by epigenetic modifications. DNA methylation operates as a very stable repressive mark in this process. In this study, we investigated the relationship between DNA methylation and expression of pluripotency-associated genes (OCT4, NANOG and SOX2), a trophectoderm (TE)-specific gene (ELF5), and genes associated with neural differentiation (SOX2 and VIMENTIN) in porcine Day 10 (E10) epiblast, hypoblast, and TE as well as in epiblast-derived neural progenitor cells (NPCs). We found that OCT4, NANOG, and SOX2 were highly expressed in the epiblast and hypoblast, while VIMENTIN was only highly expressed in the epiblast. Moreover, low expression of OCT4, NANOG, SOX2 and VIMENTIN was noted in the TE. Most CpG sites of OCT4, NANOG, SOX2 and VIMENTIN displayed low methylation levels in the epiblast and hypoblast and, strikingly, also in the TE. Hence, the expression patterns of these genes were not directly related to levels of DNA methylation in the TE in contrast to the situation in the mouse. In contrast, ELF5 was exclusively expressed in the TE and was correspondingly hypomethylated in this tissue. In NPCs, we observed down-regulation of NANOG and OCT4 expression, which correlated with hypermethylation of their promoters, whereas VIMENTIN displayed up-regulation in accordance with hypomethylation of its promoter. In conclusion, DNA methylation is an inconsistently operating epigenetic mechanism in porcine E10 blastocysts, whereas in porcine epiblast-derived NPCs, expression of pluripotency-associated and differentiation genes appear to be regulated by this modification.     

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.     

Sox15 is up regulated in the embryonic mouse testis

The mammalian sex determining region on the Y chromosome, SRY, is the founding member of the SOX gene family. SOX genes share a common DNA-binding motif termed the HMG box and have diverse roles in vertebrate embryonic development and tissue differentiation. Sox15 expression was analysed during mouse embryogenesis by whole-mount in situ hybridisation and Real Time RT-PCR. Sox15 was found to be expressed in developing mouse gonads from 11.5 dpc to 13.5 dpc with a peak of expression at 12.5 dpc. Expression was approximately twice as high in the male gonad as in the female gonad.     

PAUPAR and PAX6 sequentially regulate human embryonic stem cell cortical differentiation

Long noncoding RNAs (lncRNAs) play a wide range of roles in the epigenetic regulation of crucial biological processes, but the functions of lncRNAs in cortical development are poorly understood. Using human embryonic stem cell (hESC)-based 2D neural differentiation approach and 3D cerebral organoid system, we identified that the lncRNA PAUPAR, which is adjacent to PAX6, plays essential roles in cortical differentiation by interacting with PAX6 to regulate the expression of a large number of neural genes. Mechanistic studies showed that PAUPAR confers PAX6 proper binding sites on the target neural genes by directly binding the genomic regions of these genes. Moreover, PAX6 recruits the histone methyltransferase NSD1 through its C-terminal PST enrichment domain, then regulate H3K36 methylation and the expression of target genes. Collectively, our data reveal that the PAUPAR/PAX6/NSD1 complex plays a critical role in the epigenetic regulation of hESC cortical differentiation and highlight the importance of PAUPAR as an intrinsic regulator of cortical differentiation.     

Generation of Induced Pluripotent Stem Cells in Rabbits: POTENTIAL EXPERIMENTAL MODELS FOR HUMAN REGENERATIVE MEDICINE*

Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2, and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of basic FGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4, and NANOG, whereas the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm, and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem cell counterparts we generated recently. However, their global gene expression analysis revealed a slight but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and embryonic stem cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans.     

Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research as well as a potential cell resource for therapy. However, each hES cell line demonstrates different identity. It is desirable to obtain more fully characterized hES cell lines with newly developed technologies associated with hES cell culture. Here, we report our experience of efficient derivation of three new Chinese hES cell lines (SHhES2, SHhES3, and SHhES4) from in vitro fertilization discarded embryos donated by women with polycystic ovary syndrome. These cell lines were derived under conditions minimizing exposure to animal components and maintained at an undifferentiated state for long-term culture. They retained a normal karyotype and expressed ALP, OCT4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. RT-PCR analysis also revealed high expression levels of pluripotency markers such as OCT4, LEFTY A, SOX2, TDGF-1, THY1, FGF4, NANOG, and REX1. When suspended in low-attachment culture dishes, embryoid bodies formed and were comprised of various differentiated cell types from all three embryonic germ layers. However, well-shaped teratomas were only harvested from line SHhES2, not from SHhES3 and SHhES4, indicating that the differentiation ability in vivo differs among the three cell lines. Collectively, the three new hES cell lines were established and fully characterized. The effort paves the way toward generating hES cell lines without contamination by animal components. All of these cell lines are available by contact Ying Jin at yjin@sibs.ac.cn.     

Caffeine treatment of ovine cytoplasts regulates gene expression and foetal development of embryos produced by somatic cell nuclear transfer

Treatment of ovine oocytes during the latter stages of maturation in vitro with caffeine, a phosphodiesterase inhibitor, can increase the activities of maturation promoting factor and mitogen‐activated protein kinases at metaphase II. When used as cytoplast recipients for somatic cell nuclear transfer (NT), caffeine‐treated oocytes produced blastocysts with increased cell numbers. The objectives of these studies were to determine the effects of caffeine treatment on the expression profile of genes involved in early embryonic development and whether induction or maintenance of pregnancy was subsequently altered. No differences in overall expression patterns were observed between fertilised, caffeine‐treated fertilised and parthenogenetic embryos. In control NT embryos, altered levels of gene expression were found for OCT4, five genes regulated by OCT4 (H2AF.Z, NANOG, SOX2, FGF4 and INFT) and the heat‐shock response genes (HSP27 and HSP70.1). Levels of OCT4, H2AF.Z, NANOG, HSP 27 and FGF4 decreased, while those of INFT, HSP70.1 and SOX2 increased. In contrast, expression levels of these genes in caffeine‐treated NT embryos were similar to those in fertilised controls. Following transfer to surrogate recipients no differences were observed in the frequency of pregnancy; however, ewes receiving caffeine‐treated embryos maintained pregnancies for longer periods and delivered a live lamb. Taken together, these results suggest that treatment of ovine oocytes with caffeine can affect gene expression and improve developmental competence. Further studies on the mechanisms behind this alteration of gene expression are required and will aid in understanding the molecular mechanisms involved in nuclear reprogramming. Mol. Reprod. Dev. 77:876–887, 2010. © 2010 Wiley‐Liss, Inc.     

DNMT3B inhibits the re-expression of genes associated with induced pluripotency

DNMT3B is a de novo DNA methyltransferase that is highly expressed in mouse and human embryonic stem (ES) cells and has been shown to be essential for differentiation of mouse ES cells toward different lineages. In the present study, we found that DNMT3B is rapidly down-regulated in human ES cells during retinoic acid (RA)-induced differentiation compared with DNMT3A2, which is also highly expressed in ES cells. Silencing of DNMT3B in human ES cells by an inducible shRNAi system leads to a reduction of clonal ability of the stem cells, while expression of OCT4 and NANOG is unchanged. By contrast, the germline-specific genes VASA and SCP3 and the surface antigen BE12 are down regulated following DNMT3B knockdown. Upon retinoic acid-induced differentiation, we found that depletion of DNMT3B leads to a decrease in expression of the surface antigen A2B5 and of neural tube-associated genes PAX7 and BRN3A. Consistent with its importance in stem cell differentiation, we observed that silencing of DNMT3B facilitates the generation of cells that bear the hallmarks of pluripotency. Our findings suggest a role of DNMT3B in controlling the differentiation of human ES cells and in the generation of iPS cells.     

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.