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A virus-free poly-promoter vector induces pluripotency in quiescent bovine cells under chemically defined conditions of dual kinase inhibition
- Source :
- Scopus, RUO. Repositorio Institucional de la Universidad de Oviedo, instname, PLoS ONE, Vol 6, Iss 9, p e24501 (2011), PLoS ONE
- Publication Year :
- 2011
- Publisher :
- Public Library of Science, 2011.
-
Abstract
- Authentic induced pluripotent stem cells (iPSCs), capable of giving rise to all cell types of an adult animal, are currently only available in mouse. Here, we report the first generation of bovine iPSC-like cells following transfection with a novel virus-free poly-promoter vector. This vector contains the bovine cDNAs for OCT4, SOX2, KLF4 and c-MYC, each controlled by its own independent promoter. Bovine fibroblasts were cultured without feeders in a chemically defined medium containing leukaemia inhibitory factor (LIF) and inhibitors of MEK1/2 and glycogen synthase kinase-3 signaling (‘2i’). Non-invasive real-time kinetic profiling revealed a different response of bovine vs human and mouse cells to culture in 2i/LIF. In bovine, 2i was necessary and sufficient to induce the appearance of tightly packed alkaline phosphatase-positive iPSC-like colonies. These colonies formed in the absence of DNA synthesis and did not expand after passaging. Following transfection, non-proliferative primary colonies expressed discriminatory markers of pluripotency, including endogenous iPSC factors, CDH1, DPPA3, NANOG, SOCS3, ZFP42, telomerase activity, Tra-1-60/81 and SSEA-3/4, but not SSEA-1. This indicates that they had initiated a self-sustaining pluripotency programme. Bovine iPSC-like cells maintained a normal karyotype and differentiated into derivatives of all three germ layers in vitro and in teratomas. Our study demonstrates that conversion into induced pluripotency can occur in quiescent cells, following a previously undescribed route of direct cell reprogramming. This identifies a major species-specific barrier for generating iPSCs and provides a chemically defined screening platform for factors that induce proliferation and maintain pluripotency of embryo-derived pluripotent stem cells in livestock.
- Subjects :
- Homeobox protein NANOG
Male
Rex1
Cellular differentiation
Agricultural Biotechnology
Genetic Vectors
Induced Pluripotent Stem Cells
lcsh:Medicine
Biology
Leukemia Inhibitory Factor
Cell Line
Kruppel-Like Factor 4
Mice
SOX2
Molecular Cell Biology
Animals
Humans
Induced pluripotent stem cell
Promoter Regions, Genetic
lcsh:Science
Protein Kinase Inhibitors
Multidisciplinary
Genetically Modified Organisms
Stem Cells
lcsh:R
Teratoma
Cell Differentiation
Agriculture
Molecular biology
Chemically defined medium
Cell culture
embryonic structures
Cattle
Female
lcsh:Q
Cellular Types
Reprogramming
Biomarkers
Research Article
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Scopus, RUO. Repositorio Institucional de la Universidad de Oviedo, instname, PLoS ONE, Vol 6, Iss 9, p e24501 (2011), PLoS ONE
- Accession number :
- edsair.doi.dedup.....6343cfbbbc21230887bf31ba9b22cb6a