Madhusmita Dhupal(prof Hyun)

Information about Madhusmita Dhupal(prof Hyun)

Published on July 17, 2014

Author: dhupal

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Generation of Naive-Like Porcine-Induced Pluripotent Stem Cells Capable of Contributing to Embryonic and Fetal Development: Generation of Naive-Like Porcine-Induced Pluripotent Stem Cells Capable of Contributing to Embryonic and Fetal Development Shuh-hei Fujishiro,1 Kazuaki Nakano,Shinya Yamanaka 8/10/13 prof. Hyun 1 Stem Cells Dev. 2013 February 1; 22(3): 473–482. Published online 2012 August 13. doi : 10.1089/scd.2012.0173 PMCID: PMC3549629 Madhusmita Dhupal . Student-ID:-2013139002 PhD:1 st semester Molecular Embryology Lab Department of Animal Science CBNU Contents: Introduction and Rationale Hypothesis Objective Material & Methods Results Summary & Conclusion 8/10/13 prof. Hyun Contents I. Introduction and Rationale : Pluripotent stem cells are 2 types: -Naïve and Primed. Ontogenetically, naïve PSCs corresponds to ICM and primed PSC to epiblast . Characteristics of naïve PSCs: -Develop chimeras when injected to allogenic embryos -Dome like colony -Dependency on p-LIF -Active X-chromosome ( XaXa ) -MHC-I negative - Characteristic gene expression profile. 8/10/13 prof. Hyun 3 I. Introduction and Rationale II. Hypothesis : To convert primed PSCs into Naïve PSCs . -to check effect of p-LIF in culture media -to check 2 signal transducer inhibitors effect on conversion of primed to naïve in porcine and in human PSCs. -to develop macroscopic fluoroscent chimeras in-vitro and in-vivo. - reprogramming status during conversion of primed to naïve state. 8/10/13 prof. Hyun 4 II. Hypothesis III.Objective : Isolation of Naïve porcine iPSCs similar to mouse Naïve PSCs and in addition they must have capacity to contribute fetal developement ,. 8/10/13 prof. Hyun 5 III.Objective Injected into morulae Cultured Naïve iPSCs Hatching blasomere into uterus of pig hu -KO flouroscence gene expessing Naïve iPSCs into chimeric adult IV. Material & Methods: 8/10/13 prof. Hyun 6 IV. Material & Methods Immunohisto chemestry Flow Cytometry q-PCR RT-PCR Bisulphid sequencing electrophoresis V.RESULT: Fig.1.Naive and primed pluripotent stem cells: 8/10/13 prof. Hyun 7 V.RESULT: Fig.1.Naive and primed pluripotent stem cells Fig.2. Putative porcine iPSCs are LIF-dependent and MHC-Inegative: 8/10/13 prof. Hyun 8 Fig.2. Putative porcine iPSCs are LIF-dependent and MHC- Inegative Fig.3. Putative naive porcine iPSCs have XaXa and distinct profiles of gene expression: 8/10/13 prof. Hyun 9 Fig.3. Putative naive porcine iPSCs have XaXa and distinct profiles of gene expression Fig.4. Injection of porcine iPSCs into morulae and their in vitro development: 8/10/13 prof. Hyun 10 Fig.4. Injection of porcine iPSCs into morulae and their in vitro development Table 1. Injection of Porcine-Induced Pluripotent Stem Cells into Morulae and Their In Vitro Development :       In vitro development of embryos         No. of fluorescence-positive blastocysts (%) Host embryos Donor cells (fluorescence marker) No. of embryos injected No. of blastocysts developed (%) No. contributed to ICM No. contributed to both ICM and TE Parthenogenetic Morulae Porco Rosso iPSCs (huKO) 19 17 (89.5) 6 (35.3) 3 (17.6)   Porco Clawn Verde iPSCs (EGFP) 10 7 (70.0) 5 (71.4) 0 (0.0)   Subtotal 29 24 (82.8) 11 (45.8) 3 (12.5)   ICM (DiI) 21 20 (95.2) 17 (85.0) 16 (80.0) 8/10/13 prof. Hyun 11 Table 1. Injection of Porcine-Induced Pluripotent Stem Cells into Morulae and Their In Vitro Development Fig.5. The in utero development of porcine iPSCs. : 8/10/13 prof. Hyun 12 Fig.5. The in utero development of porcine iPSCs . Table-2: THE IN-UTERO DEVELOPMENT OF PORCINE INDUCED PLURIPOTENT STEM CELLS:       In utero development of fetuses       No. of recovered fetuses (%)   Host embryos Donor cells (fluorescence marker) No. of morulae injected Day 23 Day 65 No. of fluorescent fetuses (%) Parthenogenetic Morulae Porco Rosso or Porco Clawn Verde iPSCs (huKO or EGFP) 101 43 (42.6) — 4 (9.3)   Porco Clawn Verde iPSCs (EGFP) 43 28 (65.1) — 7 (25.0)   Subtotal 144 71 (49.3) — 11 (15.5) In Vitro Fertilized Morulae Porco Rosso iPSCs (huKO) 51 — 8 (15.7) 1 (12.5)   Porco Verde iPSCs (EGFP) 52 — 5 (9.6) 0 (0)   Subtotal 103 — 13 (12.6) 1 (7. 8/10/13 prof. Hyun 13 Table-2: THE IN-UTERO DEVELOPMENT OF PORCINE INDUCED PLURIPOTENT STEM CELLS Fig.6. The DNA-methylation status of the endogenous Oct3/4 promoter region in porcine iPSCs: 8/10/13 prof. Hyun 14 Fig.6. The DNA-methylation status of the endogenous Oct3/4 promoter region in porcine iPSCs Fig.7. The silencing of the huKO-fluorescence gene implies completely reprogrammed status.: 8/10/13 prof. Hyun 15 Fig.7. The silencing of the huKO -fluorescence gene implies completely reprogrammed status . VI: Summary & Conclusion : The putative naïve porcine iPSCs were generated using p –LIF and forskoline . It has ability to contribute fetal devlopement . P- iPSCs meet all criterion of naïvety . SSEA-1 candidate marker for naivity for mice and pig PSCs. Eras is another candidate marker for naivity for human PSCs. 8/10/13 prof. Hyun 16 VI: Summary & Conclusion Take a home massage ... : Take a home massage ... Porcine primed PSCs converted to naïve..in this way human PSCs can be investigate for conversion into naïve, which will boost to clinical theraputices . Will generate a new era of regenerative medicine. 8/10/13 prof. Hyun PowerPoint Presentation: Presented By: Madhusmita Dhupal [email protected] 8/10/13 prof. Hyun

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