Neli Ragina

Publications

• 4.15

  Impact points

  Downregulation of H19 Improves the Differentiation Potential of Mouse Parthenogenetic Embryonic Stem Cells.

  Neli P Ragina, Karianne Schlosser, Jason G Knott, Patricia K Senagore, Pamela J Swiatek, Eun Ah Chang, Walid D Fakhouri, Brian C Schutte, Matti Kiupel, Jose B Cibelli

  Stem cells and development. 07/2011;

  Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental em... [more] Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.

• Down-regulation of H19 improves the differentiation potential of mouse parthenogenetic embryonic stem cells

  Ragina NP, Schlosser K, Knott JG, Senagore PK, Swiatek PJ, Chang EA, Fakhouri WD, Schutte BG, Kiupel M, Cibelli JB

  Stem Cells Dev. 01/2011;
• 2.58

  Impact points

  Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection.

  Pablo Ross, Ramon Rodriguez, Amy Iager, Zeki Beyhan, Kai Wang, Neli Ragina, Sook-Young Yoon, Rafael Fissore, Jose Cibelli

  Reproduction (Cambridge, England). 01/2009;

  The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca2+ oscillations and oocyte activation. During b... [more] The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca2+ oscillations and oocyte activation. During bovine somatic cell nuclear transfer (SCNT), oocyte activation is artificially achieved by combined chemical treatments that induce a monotonic rise in intracellular Ca2+ and inhibit either phosphorylation or protein synthesis. In this study, we tested the hypothesis that activation of bovine nuclear transfer embryos by PLCZ improves nuclear reprogramming. Injection of PLCZ cRNA into bovine SCNT units induced Ca2+ oscillations similar to those observed after fertilization and supported high rates of blastocyst development similar to that seen in embryos produced by IVF. Furthermore, gene expression analysis at the 8-cell and blastocyst stages revealed a similar expression pattern for a number of genes in both groups of embryos. Lastly, levels of trimethylated lysine 27 at histone H3 in blastocysts were higher in bovine nuclear transfer embryos activated using CHX and DMAP than in those activated using PLCZ or derived from IVF. These results demonstrate that exogenous PLCZ can be used to activate bovine SCNT-derived embryos and support the hypothesis that a fertilization-like activation response can enhance some aspects of nuclear reprogramming.

• Parthenogenetic Embryonic Stem Cells in Nonhuman Primates

  Neli Petrova Ragina, Jose Bernardo Cibelli

  Trends in Stem Cell Biology and Technology. 01/2009;
• 2.58

  Impact points

  Polycomb genes expression and histone H3 lysine 27 tri-methylation changes during bovine preimplantation development.

  Pablo Ross, Neli Ragina, Ramon Rodriguez, Amy Iager, Kannika Siripattarapravat, Nestor Lopez-Corrales, Jose Cibelli

  Reproduction (Cambridge, England). 10/2008;

  Tri-methylation of Histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes and the dynamics of H3K27me3 during bovine oocyte maturation and preimpl... [more] Tri-methylation of Histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes and the dynamics of H3K27me3 during bovine oocyte maturation and preimplantation development. Oocytes and in vitro produced embryos were collected at different stages of development. Polycomb genes expression was analyzed by real-time quantitative RT-PCR and immunofluorescence. Global H3K27me3 levels were determined by semiquantitative immunofluorescence. Transcripts for EZH2, EED and SUZ12 were detected at all stages analyzed, with EZH2 levels being highest of the three at early stages of development. By the time the embryo reached the blastocyst stage the level of PcG gene mRNA levels significantly increased. Immunofluorescence staining indicated nuclear expression of EZH2 at all stages while nuclear localized EED and SUZ12 were only evident at morula and blastocyst stages. Semiquantitative analysis of H3K27me3 levels showed that nuclear fluorescence intensity was highest in immature oocytes, steadily decreased after fertilization to reach a nadir at the 8-cell stage, and then increased at the blastocyst stage. These results suggest that the absence of polycomb repressive complex 2 proteins localized to the nucleus of early embryos could be responsible for the gradual decrease in H3K27me3 during early preimplantation development.
• 2.69

  Impact points

  Trichostatin A Improves Histone Acetylation in Bovine Somatic Cell Nuclear Transfer Early Embryos.

  Amy E Iager, Neli P Ragina, Pablo J Ross, Zeki Beyhan, Kerrianne Cunniff, Ramon M Rodriguez, Jose B Cibelli

  Cloning and stem cells. 04/2008;

  Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin ... [more] Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.