Abstract
Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.
Highlights
While embryonic stem cells (ESCs) can be derived from mouse and human blastocysts, ESCs from farm animals have not yet been established
We evaluated whether cell cycle synchronization into G1 could improve preimplantation development of nuclear transfer (NT) embryos using ovine induced pluripotent stem cells (iPSCs) compared to parental fetal fibroblasts (PFFs)
Characterization of bFGF/activin-dependent ovine iPSCs The ovine iPSCs used in this study were generated by forced overexpression of the four Yamanaka factors (Oct4, Sox2, c-Myc and Klf4) in ovine foetal fibroblasts using retroviral transduction
Summary
While embryonic stem cells (ESCs) can be derived from mouse and human blastocysts, ESCs from farm animals have not yet been established. IPSCs have been used to produce cloned mice by NT with equivalent success to ESC donors (Kou et al, 2010; Zhou et al, 2010), a large joint effort failed to produce cloned piglets using porcine iPSCs and successful term development was only obtained when iPSCs were differentiated before using them as NT donors (Fan et al, 2013) It is not clear what prevented postimplantation development of embryos reconstructed with undifferentiated iPSCs, but it is possible that either the lack of cell cycle synchronization prior to NT or the quality of the iPSCs compromised the developmental competence of those embryos. Some reports show that G2/M donors can be used (Lai et al, 2002; Lai et al, 2001), other reports have found variable levels of aneuploidy and compromised development of the resulting reconstructed embryos (Alberio et al, 2000; Yuan et al, 2014; Zhang et al, 2004)
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