title: Generation and characterization of reprogrammed sheep induced pluripotent stem cells.
abstract: Embryonic stem cells (ESCs) from domestic species have numerous potential applications in agricultural and biomedical sciences; however, despite intensive efforts, derivation of ESCs from sheep remains elusive. The objective was to derive sheep induced pluripotent stem cells (iPSCs), as an alternative pluripotent cell type to ESCs, from sheep fibroblasts by ectopic expression of heterologous transcription factors OCT4, SOX2, KLF4, and cMYC. Sheep fibroblasts were infected with pantropic retroviruses coding the four transcription factors and reprogrammed to pluripotency at a rate of 0.002%. The sheep iPSCs (siPSCs) reactivated endogenous OCT4 and SOX2 genes assessed by qRT-PCR and immuno-cytochemistry, retained normal karyotyping, and more importantly, concurrently silenced all exogenous transgenes. The siPSCs were enzymatically dissociated to single cells, making them amenable to efficient transfection and fluorescent-activated cell sorting techniques. Further, the siPSCs differentiated in vitro to form embryoid bodies, and in vivo to form robust teratomas, containing cells representative of the three germ layers. Moreover, when injected into diploid or tetraploid sheep embryos, siPSCs contributed to the inner cell mass of resulting blastocysts, suggesting true pluripotential. These reprogrammed siPSCs may constitute a robust pluripotent alternative to elusive sheep ESCs, with great potential for use in agriculture and pharmaceutical biotechnology.
authors.affiliation: Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton, VIC 3168, Australia.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Deepashree
authors.initials: D
authors.lastname: Balehosur
authors.forename: Belinda
authors.initials: B
authors.lastname: Murray
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Kelly
authors.forename: Huseyin
authors.initials: H
authors.lastname: Sumer
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Verma
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D053595:Embryonic Stem Cells; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D016259:Genes, myc / Q000235:genetics; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics; D012756:Sheep; D014162:Transfection
chemical_list: D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors