Somatic Cell Nuclear Reprogramming Research Articles

Genome stabilization by RAD51-stimulatory compound 1 enhances efficiency of somatic cell nuclear transfer-mediated reprogramming and full-term development of cloned mouse embryos.

ObjectivesThe genetic instability and DNA damage arise during transcription factor‐mediated reprogramming of somatic cells, and its efficiency may be reduced due to abnormal chromatin remodelling. The efficiency in somatic cell nuclear transfer (SCNT)‐mediated reprogramming is also very low, and it is caused by development arrest of most reconstituted embryos.Materials and MethodsWhether the repair of genetic instability or double‐strand breaks (DSBs) during SCNT reprogramming may play an important role in embryonic development, we observed and analysed the effect of Rad 51, a key modulator of DNA damage response (DDR) in SCNT‐derived embryos.ResultsHere, we observed that the activity of Rad 51 is lower in SCNT eggs than in conventional IVF and found a significantly lower level of DSBs in SCNT embryos during reprogramming. To address this difference, supplementation with RS‐1, an activator of Rad51, during the activation of SCNT embryos can increase RAD51 expression and DSB foci and thereby increased the efficiency of SCNT reprogramming. Through subsequent single‐cell RNA‐seq analysis, we observed the reactivation of a large number of genes that were not expressed in SCNT‐2‐cell embryos by the upregulation of DDR, which may be related to overcoming the developmental block. Additionally, there may be an independent pathway involving histone demethylase that can reduce reprograming‐resistance regions.ConclusionsThis technology can contribute to the production of comparable cell sources for regenerative medicine.

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming.

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming.

Germ cell transplantation in pigs - advances and applications

Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice results in donor-derived spermatogenesis and transmission of the donor haplotype to offspring of recipient animals. In the pig, germ cells can be transplanted to a recipient testis by ultrasoundguided cannulation of the rete testis with delivery of cells by gravity flow. It is important to note that germ cell transplantation was successful between unrelated, immuno-competent pigs, whereas transplantation in rodents requires syngeneic or immuno-compromised recipients. Efficiency of colonization of the recipient testis by donor-derived germ cells can be improved by pretreatment of the recipient animal to deplete endogenous germ cells. Genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in production of transgenic sperm. Transgenesis through the male germ line has tremendous potential in species like pigs where embryonic stem cell technology is not available and current options to generate transgenic animals are inefficient. Introduction of a genetic change prior to fertilization will circumvent problems associated with manipulation of early embryos and developmental abnormalities associated with somatic cell nuclear transfer and reprogramming. Viral transduction of germ cells prior to transplantation has been used to generate transgenic rodents and has also shown early promising results in pigs. Current research is directed toward improving protocols for isolation and culture of porcine male germ cells to increase efficiency of transgene transmission and to allow for gene targeting prior to germ cell transplantation. It is expected that germ cell transplantation will then provide a viable alternate approach to generate germ line transgenic pigs.

Transcription Factor-Directed Re-wiring of Chromatin Architecture for Somatic Cell Nuclear Reprogramming toward trans-Differentiation.

MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation.

Transcriptional defects and reprogramming barriers in somatic cell nuclear reprogramming as revealed by single-embryo RNA sequencing

BackgroundNuclear reprogramming reinstates totipotency or pluripotency in somatic cells by changing their gene transcription profile. This technology is widely used in medicine, animal husbandry and other industries. However, certain deficiencies severely restrict the applications of this technology.ResultsUsing single-embryo RNA-seq, our study provides complete transcriptome blueprints of embryos generated by cumulus cell (CC) donor nuclear transfer (NT), embryos generated by mouse embryonic fibroblast (MEF) donor NT and in vivo embryos at each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst). According to the results from further analyses, NT embryos exhibit RNA processing and translation initiation defects during the zygotic genome activation (ZGA) period, and protein kinase activity and protein phosphorylation are defective during blastocyst formation. Two thousand three constant genes are not able to be reprogrammed in CCs and MEFs. Among these constant genes, 136 genes are continuously mis-transcribed throughout all developmental stages. These 136 differential genes may be reprogramming barrier genes (RBGs) and more studies are needed to identify.ConclusionsThese embryonic transcriptome blueprints provide new data for further mechanistic studies of somatic nuclear reprogramming. These findings may improve the efficiency of somatic cell nuclear transfer.

Factors Affecting Mouse Somatic Cell Nuclear Reprogramming by Rabbit Ooplasms.

Successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos depends on compatibilities between ooplasmic and nuclear components. However, the mechanisms by which the compatibilities are regulated are still unknown. In this study, using mouse Oct4-green fluorescent protein (GFP) cells as donors and rabbit oocytes as recipients, we show that Oct4 and other pluripotency related genes were reactivated in some of mouse-rabbit iSCNT embryos, which could also activate Oct4 promoter-driven GFP reporter gene expression. Series nuclear transfer improved the efficiency of Oct4 reactivation. DNA demethylation of Oct4 promoter was detected in GFP positive iSCNT blastocysts, whereas GFP negative iSCNT embryos showed a low efficiency. Our results demonstrate that Oct4-GFP can well label the embryos with epigenetic remodeling and reactivation of pluripotent gene expression. Abundant rabbit mitochondria specific DNAs were identified in reconstructed mouse-rabbit embryos throughout preimplantation stages. Our data demonstrate that epigenetic remodeling and the complete mitochondrial match are not necessary for successful iSCNT embryo development before implantation.

Editorial: Cell Fate.

The complexity and plasticity of cell fate determination has intrigued cell and developmental biologists for decades. Cellular differentiation is the acquisition of specialized characteristics; which is intimately associated with changes in gene expression, alterations of chromatin, and changes in nuclear architecture. Differentiating tissues exhibit a progressive restriction of cellular plasticity. However, the regenerative ability of some organisms has revealed an amazing capacity for dramatic switches in cell fate, through trans-differentiation and de-differentiation (Sanchez Alvarado and Tsonis, 2006). Furthermore, the groundbreaking work on somatic cell nuclear reprogramming and induced pluripotency has revealed that commitment to cell fate can be far more flexible than previously thought (Lensch and Mummery, 2013). In this research topic on cell fate we aimed to highlight new developments and outstanding questions in our understanding of how chromatin dynamics impact cell fate and cellular reprogramming. We include articles discussing cell fate decisions in a wide variety of contexts and model organisms. The contributions to this topic include review articles, mini-reviews, original research, and perspectives. The work described here encompasses organisms ranging from C. elegans to humans and deals with global cell fate issues of sex determination (Lau and Csankovszki), lineage choice (Chin), preventing premature differentiation (Foret et al.) cell fate and cell cycle regulation (Oyama et al.; Julian and Blais; Ma et al.; Parker), nuclear architecture (Talamas and Capelson) and how dynamic transcriptional repressors promote cell fate choices (Kok and Arnosti). We thank the authors, reviewers and editors for contributing to the stimulating discussion of the open questions in this rapidly changing field.

BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

Abstract 12444: Toll-like Receptor 3 is Required for Neonatal Heart Repair and Regeneration Following Myocardial Infarction via Glycolysis Dependent Yap/taz Activation

Regenerative therapies for repair of the damaged heart could benefit millions of patients annually. The neonatal heart has the capability of repairing/regenerating damaged myocardium. However, this capability is lost when cardiomyocyte metabolism changes from glycolysis to oxidative phosphorylation, within 7 days after birth. Interestingly, Toll-like receptors (TLRs) are required for somatic cell nuclear reprogramming, which is important for tissue regeneration. The Hippo pathway effector YAP promotes cardiac regeneration. We have reported that TLR ligands can induce protection against myocardial ischemic injury. We hypothesized that TLRs are required for cardiac regeneration in neonatal heart via glycolysis dependent YAP/TAZ activation. To evaluate our hypothesis, myocardial infarction (MI) was induced in one day of wild type (WT) and TLR3 deficient (TLR3 -/- ) neonatal mice (n=5/group). Cardiac function was examined by echocardiography 3 weeks after MI. Cardiomyocyte regeneration and proliferation were examined by histone3 phosphorylation (p-histone3) and EdU incorporation into the cardiomyocytes. In WT neonatal MI mice, cardiac function completely recovered, infarct size was smaller, more p-histone3, EdU was incorporated into the cardiomyocytes, and YAP/TAZ activation was significantly increased following MI. In contrast, TLR3 -/- neonatal hearts showed impaired cardiac function, larger infarct size, less p-histone3 and EdU incorporation and inactivation of YAP/TAZ after MI. To examine whether stimulation of TLR3 will promote glycolysis, cardiomyocytes were isolated from one day old WT neonatal mice and treated with Poly I:C, the TLR3 ligand. Poly I:C treatment significantly increases lactate production, NAD + /NADH ratio, extracellular acidification (ECAR) and YAP/TAZ activation. However, inhibition of glycolysis with 2-Deoxyglucose (2DG) prevented Poly I:C-induced neonatal cardiomyocyte proliferation and regeneration as well as YAP/TAZ activation. In vivo treatment of WT neonatal mice with 2DG (n=6/group) abolished cardiac functional recovery 3 weeks after MI. We conclude that TLR3 is required for neonatal heart regeneration and repair after MI. The mechanisms involve glycolysis dependent activation of YAP/TAZ.

Oocyte Factors Suppress Mitochondrial Polynucleotide Phosphorylase to Remodel the Metabolome and Enhance Reprogramming.

Oocyte factors not only drive somatic cell nuclear transfer reprogramming but also augment the efficiency and quality of induced pluripotent stem cell (iPSC) reprogramming. Here, we show that the oocyte-enriched factors Tcl1 and Tcl1b1 significantly enhance reprogramming efficiency. Clonal analysis of pluripotency biomarkers further show that the Tcl1 oocyte factors improve the quality of reprogramming. Mechanistically, we find that the enhancement effect of Tcl1b1 depends on Akt, one of its putative targets. In contrast, Tcl1 suppresses the mitochondrial polynucleotide phosphorylase (PnPase) to promote reprogramming. Knockdown of PnPase rescues the inhibitory effect from Tcl1 knockdown during reprogramming, whereas PnPase overexpression abrogates the enhancement from Tcl1 overexpression. We further demonstrate that Tcl1 suppresses PnPase's mitochondrial localization to inhibit mitochondrial biogenesis and oxidation phosphorylation, thus remodeling the metabolome. Hence, we identified the Tcl1-PnPase pathway as a critical mitochondrial switch during reprogramming.

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The ChariotTM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.

Panning for Long Noncoding RNAs to Improve Somatic Cell Nuclear Transfer Reprogramming Efficiency: Challenges and Opportunities

Since the cloned sheep “Dolly” was born in 1997, somatic cell nuclear transfer (SCNT) has been achieved in many species, however, to date, the overall cloning efficiency is still low, and this limits the large-scale application in basic research, agriculture and medicine, etc. It is generally believed that low cloning efficiency is mainly due to aberrant epigenetic reprogramming. In cloned embryos, DNA methylation, histone modifications and genomic imprinting, etc, are usually disrupted, and these incomplete epigenetic reprogramming causes continuous expression of tissue specific genes, no effective activation of genes related to early embryo development, and aberrant transcription of imprinted genes, etc, thereby leading to poor cloning efficiency.

Mitosis gives a brief window of opportunity for a change in gene transcription.

Author SummaryCells are dividing very actively at a time in development when new gene expression and new cell lineages arise. At mitosis, most transcription factors are temporarily displaced from chromosomes. We show that, after transplantation to oocytes, somatic cell nuclei that have been synchronized in mitosis can be reprogrammed to pluripotency gene expression up to 100 times faster than interphase nuclei. We find that, as cells traverse mitosis, their genes pass through a temporary phase of unusually high responsiveness to oocyte reprogramming factors (mitotic advantage). Many other genes in the genome have also shown a mitotic advantage, which affects the rate rather than the final level of transcriptional enhancement. This is attributable to a chromatin state rather than to more rapid passage of reprogramming factors through the nuclear membrane. Histone H2A deubiquitination at mitosis is required for the acquisition of mitotic advantage. Our results support the general principle that a temporary access of cytoplasmic factors to genes during mitosis facilitates somatic cell nuclear reprogramming and the acquisition of new cell fates in normal development.

Therapeutic potential of somatic cell nuclear transfer for degenerative disease caused by mitochondrial DNA mutations.

Induced pluripotent stem cells (iPSCs) hold much promise in the quest for personalised cell therapies. However, the persistence of founder cell mitochondrial DNA (mtDNA) mutations limits the potential of iPSCs in the development of treatments for mtDNA disease. This problem may be overcome by using oocytes containing healthy mtDNA, to induce somatic cell nuclear reprogramming. However, the extent to which somatic cell mtDNA persists following fusion with human oocytes is unknown. Here we show that human nuclear transfer (NT) embryos contain very low levels of somatic cell mtDNA. In light of a recent report that embryonic stem cells can be derived from human NT embryos, our results highlight the therapeutic potential of NT for mtDNA disease, and underscore the importance of using human oocytes to pursue this goal.

Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer: a review

Oocytes are unique cells with the inherent capability to reprogram nuclei. The reprogramming of the somatic nucleus from its original cellular state to a totipotent state is essential for term development after somatic cell nuclear transfer. The nuclear-associated factors contained within oocytes are critical for normal fertilization by sperm or for somatic cell nuclear reprogramming. The chromatin of somatic nuclei can be reprogrammed by factors in the egg cytoplasm whose natural function is to reprogram sperm chromatin. The oocyte first obtains its reprogramming capability in the early fetal follicle, and then its capacity is enriched in the late growth phase and reaches its highest capability for reprogramming as fully-grown germinal vesicle oocytes. The cytoplasmic milieu most likely contains all of the specific transcription and/or reprogramming factors neces- sary for cellular reprogramming. Certain transcription factors in the cytoplast may be critical as has been demonstrated for induced pluripotent stem cells. The maternal pronucleus exerts a predominant, transcription- dependent effect on embryo cytofragmentation, with a lesser effect imposed by the ooplasm and the paternal pronucleus. With deep analysis of transcriptomics in oocytes and early developmental stage embryos more maternal transcription factors inducing cellular reprogram- ming will be identified.

The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes

Sperm and eggs carry distinctive epigenetic modifications that are adjusted by reprogramming after fertilization. The paternal genome in a zygote undergoes active DNA demethylation before the first mitosis. The biological significance and mechanisms of this paternal epigenome remodelling have remained unclear. Here we report that, within mouse zygotes, oxidation of 5-methylcytosine (5mC) occurs on the paternal genome, changing 5mC into 5-hydroxymethylcytosine (5hmC). Furthermore, we demonstrate that the dioxygenase Tet3 (ref. 5) is enriched specifically in the male pronucleus. In Tet3-deficient zygotes from conditional knockout mice, paternal-genome conversion of 5mC into 5hmC fails to occur and the level of 5mC remains constant. Deficiency of Tet3 also impedes the demethylation process of the paternal Oct4 and Nanog genes and delays the subsequent activation of a paternally derived Oct4 transgene in early embryos. Female mice depleted of Tet3 in the germ line show severely reduced fecundity and their heterozygous mutant offspring lacking maternal Tet3 suffer an increased incidence of developmental failure. Oocytes lacking Tet3 also seem to have a reduced ability to reprogram the injected nuclei from somatic cells. Therefore, Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA following natural fertilization and may also contribute to somatic cell nuclear reprogramming during animal cloning.

Understanding Oxidative Stress in Donor Fibroblasts for Enhancing Reprogramming Efficiency.

The low success rate of cloning by somatic cell nuclear transfer can be attributed to the remarkable difficulty in reprogramming differentiated donor nuclei. Reports indicate this is largely due to differentiated cells having undergone epigenetic modifications, telomere shortening and oxidative stress accumulation as they age, resulting in a decreased ability to generate induced pluripotent stem cells. Cellular senescence, the irreversible loss of replicative capacity in cells, is a significant barrier to transgenesis and somatic cell nuclear reprogramming in mammals. Cellular senescence can be triggered prematurely by exposure to reactive oxygen species (ROS)-induced oxidative stress. p66shc, a stress sensor that may be linked to ROS-induced senescence, has been suggested to trigger apoptotic responses by generating mitochondrial H2O2 in response to exogenous H2O2 exposure. This interaction increases the amount of oxidative stress experienced within a cell and may contribute to the difficulties of nuclear reprogramming differentiated somatic cells. The purpose of this study was to examine the role of p66Shc in the ROS-mediated somatic cell senescence response. Bovine fibroblasts were treated for two hours with H2O2 concentrations 25 micromol/L, 50 micromol/L, 100 micromol/L, 150 micromol/L and 200 micromol/L in order to determine the effects of ROS on the expression of p66Shc and on the induction of the senescence signaling pathway. Post H2O2 treatment, cells were treated with pharmacological inhibitor Juglone to modulate p66shc activity by inhibiting its translocation into the mitochondria via peptidly propyl isomerase-1 (PIN1). Bongkrekic Acid and Difumarate, pharmacological inhibitors that inhibit the opening of mitochondrial permeability transition pores (MPTP), and interfere with mitochondrial membrane potential, respectively, were used to prevent the subsequent release of mitochondrial ROS into the intracellular environment. Following treatment with pharmacological inhibitors, the number of floating dead cells was quantified using a hemocytometer. Cells sticking to the bottom of the culture dish were then washed with fresh media and cultured for either 24 hours or 72 hours. Senescent cells were detected using a SA-beta-galactosidase staining assay. Real-time PCR was used to quantitatively assess altered expression of critical genes regulating mitochondrial ROS-mediated signaling: p66shc, PIN1, ANT3, Hsp70 and COX. Results indicate that as H2O2 dosage increases, apoptosis increases; however, SA-beta-galactosidase measured senescence decreases. This indicates cells are sensitive to rising levels of H2O2 and make a choice to enter senescence based on the oxidative insult they face. Low levels of p66shc expression are associated with senescence, while high levels are associated with apoptosis. These results indicate that the presence of exogenous H2O2 induces the modulation of p66shc expression, suggesting that p66Shc may serve as an integration point for ROS-mediated senescence signaling. Future studies would focus on alleviating senescence in bovine cultures to enhance reprogramming efficiency. Funding provided by the Canadian Institute of Health Research. (poster)

Histone variant macroH2A confers resistance to nuclear reprogramming.

How various layers of epigenetic repression restrict somatic cell nuclear reprogramming is poorly understood. The transfer of mammalian somatic cell nuclei into Xenopus oocytes induces transcriptional reprogramming of previously repressed genes. Here, we address the mechanisms that restrict reprogramming following nuclear transfer by assessing the stability of the inactive X chromosome (Xi) in different stages of inactivation. We find that the Xi of mouse post-implantation-derived epiblast stem cells (EpiSCs) can be reversed by nuclear transfer, while the Xi of differentiated or extraembryonic cells is irreversible by nuclear transfer to oocytes. After nuclear transfer, Xist RNA is lost from chromatin of the Xi. Most epigenetic marks such as DNA methylation and Polycomb-deposited H3K27me3 do not explain the differences between reversible and irreversible Xi. Resistance to reprogramming is associated with incorporation of the histone variant macroH2A, which is retained on the Xi of differentiated cells, but absent from the Xi of EpiSCs. Our results uncover the decreased stability of the Xi in EpiSCs, and highlight the importance of combinatorial epigenetic repression involving macroH2A in restricting transcriptional reprogramming by oocytes.

Somatic cell nuclear reprogramming of mouse oocytes endures beyond reproductive decline

The mammalian oocyte has the unique feature of supporting fertilization and normal development, while capable of reprogramming nuclei of somatic cells toward pluripotency, and occasionally even totipotency. While oocyte quality is known to decay with somatic aging, it is not a given that different biological functions decay concurrently. In this study, we tested whether oocyte's reprogramming ability decreases with aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT), giving rise to higher blastocysts rates compared to young donors ooplasts. Despite the differences in transcriptome between climacteric and young ooplasts, gene expression profiles of SCNT blastocysts were very similar. Importantly, embryonic stem cell lines with capacity to differentiate into tissues from all germ layers were derived from SCNT blastocysts obtained from climacteric ooplasts. Although apoptosis-related genes were down-regulated in climacteric ooplasts, and reprogramming by transcription factors (direct-induced pluripotency) benefits from the inhibition of p53-mediated apoptosis, reprogramming capacity of young ooplasts was not improved by blocking p53. However, more outgrowths were derived from SCNT blastocysts developed in the presence of a p53 inhibitor, indicating a beneficial effect on trophectoderm function. Results strongly suggest that oocyte-induced reprogramming outcome is determined by the availability and balance of intrinsic pro- and anti-reprogramming factors tightly regulated and even improved throughout aging, leading to the proposal that oocytes can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction.

Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency gene reactivation

When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocyte-specific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes.