Ell3 Enhances Differentiation of Mouse Embryonic Stem Cells by Regulating Epithelial-Mesenchymal Transition and Apoptosis

Ell3 is a testis-specific RNA polymerase II elongation factor whose cellular function is not clear. The present study shows that Ell3 is activated during the differentiation of mouse embryonic stem cells (mESCs). Furthermore, Ell3 plays a critical role in stimulating lineage differentiation of mESCs by promoting epithelial-mesenchymal transition (EMT) and suppressing apoptosis. Mouse ESCs engineered to stably express Ell3 were rapidly differentiated compared with control cells either under spontaneous differentiation or neural lineage-specific differentiation conditions. Gene expression profile and quantitative RT-PCR analysis showed that the expression of EMT markers, such as Zeb1 and Zeb2, two major genes that regulate EMT, was upregulated in Ell3-overexpressing mESCs. Remarkably, knockdown of Zeb1 attenuated the enhanced differentiation capacity of Ell3-overexpressing mESCs, which indicates that Ell3 plays a role in the induction of mESC differentiation by inducing EMT. In contrast to Ell3-overexpressing mESCs, Ell3-knock down mESCs could not differentiate under differentiation conditions and, instead, underwent caspase-dependent apoptosis. In addition, apoptosis of differentiating Ell3-knock out mESCs was associated with enhanced expression of p53. The present results suggest that Ell3 promotes the differentiation of mESCs by activating the expression of EMT-related genes and by suppressing p53 expression

Human Embryonic Stem Cells and Embryonal Carcinoma Cells Have Overlapping and Distinct Metabolic Signatures

While human embryonic stem cells (hESCs) and human embryonal carcinoma cells (hECCs) have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS). Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline) or lower (e.g., glutamic acid, mannitol, malic acid, GABA) in hESCs (H9) compared to hECCs (NTERA2), these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O2 while oxidative phosphorylation (OXPHOS) is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors

The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals

Generation of calcium oscillations in mammalian eggs: Impact on activation and development and implications for cloning

The mechanisms by which the sperm generates long lasting [Ca2+ ]i oscillations in mammalian oocytes is discussed in the first chapter. In Chapter 2, a study was undertaken to determine if injection of porcine sperm factor(s) (pSF) can be used to activate bovine oocytes during nuclear transfer. It was found that injection of 5 mg/ml pSF triggered [Ca 2+]i oscillations that resembled those associated with fertilization. This concentration of pSF supported in vitro and in vivo development up to 60–90 d of gestation. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because it was unable to support oscillations pass 3 to 5 h post-injection. Likewise, a single injection of pSF failed to trigger down-regulation of the inositol 1,4,5-trisphosphate receptor-1 sub-type (IP3R-1). These results demonstrate that pSF can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited due to the premature cessation of the induced oscillations. In chapter 3 we evaluated the temporal release of the Ca2+-active factor during mouse fertilization and its possible association with the perinuclear theca (PT) of mammalian sperm. Between 15–60 min post sperm entry a significant portion of the Ca2+ releasing activity became dissociated from the sperm head. The loss of the Ca2+ releasing activity coincided with exposure and solubilization of the sperm\u27s PT, although disassembly of the PT did not appear to be required for the release of the Ca2+ factor. Lastly, the conditions in the egg that promote release of the sperm\u27s Ca2+ factor do not appear to be cell cycle specific. Finally, in chapter 4 we evaluated the impact of the IP3R-1 on the generation of Ca2+ release in bovine oocytes. Bovine oocytes with fewer numbers of IP3R-1 were produced by injection of adenophostin A, a potent antagonist of the IP3R-1 that triggers degradation of the receptors. Western blot analysis revealed that \u3e80% of the receptors were degraded. The ability of IP3R-1 deficient oocytes to trigger Ca2+ release was examined by injecting IP3 and pSF, two agonists of the IP3R-1. IP3-induced Ca2+ release was partially blocked in IP3R-1 deficient oocytes. Moreover, injection of pSF into IP3R-1 deficient oocytes failed to establish persistent Ca2+ responses. These results suggest that IP3R-1 number has significant impact on the generation of Ca2+ release in bovine oocytes

Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals.

We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed.The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells.Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals

PPARβ Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation.

Epigenetic post-transcriptional modifications of histone tails are thought to help in coordinating gene expression during development. An epigenetic signature is set in pluripotent cells and interpreted later at the onset of differentiation. In pluripotent cells, epigenetic marks normally associated with active genes (H3K4me3) and with silent genes (H3K27me3) atypically co-occupy chromatin regions surrounding the promoters of important developmental genes. However, it is unclear how these epigenetic marks are recognized when cell differentiation starts and what precise role they play. Here, we report the essential role of the nuclear receptor peroxisome proliferator-activated receptor β (PPARβ, NR1C2) in Xenopus laevis early development. By combining loss-of-function approaches, large throughput transcript expression analysis by the mean of RNA-seq and intensive chromatin immunoprecipitation experiments, we unveil an important cooperation between epigenetic marks and PPARβ. During Xenopus laevis gastrulation PPARβ recognizes H3K27me3 marks that have been deposited earlier at the pluripotent stage to activate early differentiation genes. Thus, PPARβis the first identified transcription factor that interprets an epigenetic signature of pluripotency, in vivo, during embryonic development. This work paves the way for a better mechanistic understanding of how the activation of hundreds of genes is coordinated during early development