Exogenous human OKSM factors maintain pluripotency gene expression of bovine and porcine iPS-like cells obtained with STEMCCA delivery system

Abstract

ObjectivesThe use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated.ResultsProtocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals’ iPS cells.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3627-8) contains supplementary material, which is available to authorized users.

Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance

“…Comparing reprogramming efficiencies indicated that the less differentiated WJCs were more efficient in generating ALP‐positive iPSC‐like colony formation compared to BEFs. This observation also supported previous studies that concluded bovine fibroblasts present an epigenetic roadblock and failed to reprogram (Canizo et al, ; Kawaguchi et al, ). It was reported that bovine amnion‐derived cells (Kawaguchi et al, ), and mammary epithelial cells (Cravero et al, ), are more easily reprogrammed compared to fibroblasts.…”

“…High potential for different biotechnology applications has resulted in numerous attempts to derive bovine iPSCs (Bai et al, 2016;Canizo et al, 2018;Cao et al, 2012;Cravero et al, 2015;Han et al, 2011;Heo et al, 2015;Huang et al, 2011;Kawaguchi et al, 2015;Lin et al, 2014;Sumer et al, 2011;Talluri et al, 2015;Wang et al, 2013;Zhao et al, 2017). However, these studies mark limited success and attempt only to replicate existing techniques.…”

“…Current measures of the quality of bovine iPSCs have remained quite arbitrary. Previous studies that describe the generation of bovine iPSCs either show that exogenous transgenes are not silenced (Cao et al, 2012;Cravero et al, 2015;Han et al, 2011;Heo et al, 2015;Huang et al, 2011;Sumer et al, 2011;Talluri et al, 2015;Zhao et al, 2017), or this property was not evaluated (Canizo et al, 2018;Lin et al, 2014;Wang et al, 2013). In the case of doxycycline-inducible OKS and M genes as piggyBac transposons, continuous induction of transgene expression was required to maintain bovine iPSCs (Kawaguchi et al, 2015).…”

“…While there is little doubt that the reprogramming process across different species is conserved, there exist confounding factors underscored by the limited success in iPSC derivation from ruminant species (Maruotti et al, 2012). Although several manuscripts describe bovine iPSC derivation (Bai et al, 2016;Canizo et al, 2018;Cao et al, 2012;Cravero et al, 2015;Han et al, 2011;Heo et al, 2015;Huang et al, 2011;Kawaguchi et al, 2015;Lin et al, 2014;Sumer et al, 2011;Talluri et al, 2015;Wang et al, 2013;Zhao et al, 2017), there remain several inconsistencies, serious gaps in characterization, and inappreciable research progress toward fundamental understanding or commercial application of these cells (Ezashi, Yuan, & Roberts, 2016). Perhaps, the core problems parallel shortcomings in deriving embryonic stem cells (ESCs) from ruminants (Ezashi et al, 2016).…”

Induced pluripotent stem cells throughout the animal kingdom: Availability and applications

“…Testicular cells were induced into pluripotency after electroporation of OCT4 alone[55]. However, silencing of exogenous factors when integrated was not reported, and some studies were unable to characterize bovine iPSCs (biPSCs) after culture due to characteristics related to quiescence in vitro [56,57]. It has been shown that buffalo fetal fibroblasts can be retrovirally reprogrammed into iPSC by buffalo OSKM, and that the generation efficiency of biPSCs can be increased by inhibiting p53 expression[58] (Supplemental material 1).…”