Published on July 9, 2014
PRESENTATION:-3: PRESENTATION:-3 MADHUSMITA DHUPAL STUDENT ID:2013139002: MADHUSMITA DHUPAL STUDENT ID:2013139002 Pig Epiblast Stem Cells Depend on Activin /Nodal Signaling for Pluripotency and Self-Renewal Ramiro Alberio 1 , Nicola Croxall 1 , and Cinzia Allegrucc 2 1 . Division of Animal Sciences, School of Biosciences 2 . School of Veterinary Medicine and Science, University of Nottingham, Loughborough, United Kingdom STEM CELLS AND DEVELOPMENT Volume 19, Number 10, 2010 © Mary Ann Liebert , Inc. DOI: 10.1089/scd.2010.0012 Contents: Introduction and Rationale Hypothesis Objective Material & Methods Results Summary & Conclusion Contents Introduction and Rationale: Introduction and Rationale Activin /Nodal singalling were required for maintaining pluripotency and self renewal of mouse epiblast stem cells and human embryonic stem cells. Human embryonic stem cells ( hESC ) are also derived from blastocysts ; however, these cells depend on basic fibroblast growth factor ( bFGF ) and Activin A for pluripotency and self-renewal ( J Cell Sci. 2005) Introduction and Rationale: Introduction and Rationale Pluripotent cells derived from mouse epiblasts , a part of the ICM, also require bFGF and Activin A for pluripotency and self-renewal. ( Nature. 2007 ) Recent reports show that bFGF and Activin A are also necessary for maintaining rabbit ESC.( Exp Cell Res. 2009 ) Hypothesis: Hypothesis FGF/ Activin /Nodal is a conserved signaling pathway for maintenance of pluripotency in mammals, and this can be exploited for developing strategies for the derivation of ESC from PORCINE embryos ….??? OBJECTIVES: OBJECTIVES Epiblast isolation, stem cell cultures, and in vitro differentiation Immunocytochemistry and karyotype Signaling inhibitors Gene expression Material and Methods: Material and Methods RESULTS:: RESULTS: Results: Results FIG. 1: Isolation and culture of pig epiblasts : FIG. 1: Isolation and culture of pig epiblasts FIG. 2: Characterization of pig epiblast stem cell lines (pEpiSC). : FIG. 2: Characterization of pig epiblast stem cell lines ( pEpiSC ). FIG. 3 : Signaling pathways required for pig epiblast stem cell lines (pEpiSC) pluripotency. : FIG. 3 : Signaling pathways required for pig epiblast stem cell lines ( pEpiSC ) pluripotency . PowerPoint Presentation: FIG. 4: Differentiation potential of pig epiblast stem cell lines( pEpiSC ). FIG. 5. Differentiation to trophectoderm and germ cell lineage in response to BMP-4. : FIG. 5. Differentiation to trophectoderm and germ cell lineage in response to BMP-4. DISCUSSION AND CONCLUSION: DISCUSSION AND CONCLUSION pEpiSC express the core pluripotency factors OCT4 (or POU5F1), NANOG, SOX2, and NODAL , but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specific JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC . In contrast, cells grown with the Alk-5 inhibitor SB431542 , which blocks Activin /Nodal pathway , differentiated readily toward the neural lineage. pEpiSC are pluripotent , as established by their differentiation potential to ectoderm, mesoderm, and endoderm DISCUSSION AND CONCLUSION: DISCUSSION AND CONCLUSION pEpiSC cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). This paper show that pEpiSC can reactivate the specific germ cell markers DAZL and VASA after induction with BMP-4 . In conclusion, this paper demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin /Nodal signaling . . Take Home Message.........: Take Home Message ......... This study provides further evidence that maintenance of pluripotency via Activin /Nodal signal is conserved in mammals. Further experiments should be focused at establishing the identity of the cells in the pig epiblast . Further work is needed to establish the proper differentiation conditions for pEpiSC into lineages.