Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors

Abstract

Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…iPSCs were first derived from mouse fibroblasts by overexpression of four reprogramming factors-Oct4, Sox2, Klf4, and c-Myc-and subsequently from human fibroblasts by overexpression of OCT4, SOX2, LIN28, and NANOG (Takahashi and Yamanaka, 2006;Yu et al, 2007) after retroviral transfection. Retroviral and lentiviral delivery of core reprogramming factors allowed the derivation of iPSCs from several other mammals, including rat (Liao et al, 2009), dog (Shimada et al, 2010), pig (Esteban et al, 2009), rhesus monkey (Liu et al, 2008), sheep (Bao et al, 2011), goat , horse (Nagy et al, 2011), cattle (Han et al, 2011), buffalo (Deng et al, 2012), and of iPS-like cells from nonmammalian vertebrates (Rossello et al, 2013). iPSCs derived from somatic tissue are particularly promising in farm animals in which true, germline-competent embryonic stem cells (ESCs) could not yet be derived (Kumar et al, 2015).…”

Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

“…Murine fibroblasts were reprogrammed into so-called induced pluripotent stem (iPS) cells a couple of years ago [1]. Meanwhile iPS cells have successfully been established from other species including humans [2], rats [3], monkeys [4], dogs [5], sheep [6], and pigs [7].…”

A poor imitation of a natural process

“…However, simple, straightforward assays are also widely used; some of these assays are not considered to be precise or qualified, such as colony appearance, alkaline phosphatase (AP) staining or Nanog expression. The use of such assays leads to the identification of false positives and the incorrect evaluation as human, 47 sheep 48 and goat. 49 However, these factors are potent viral oncoproteins that can inactivate the p53 and the retinoblastoma pathways.…”