Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer

German, Sergio D.; Campbell, Keith H.S.; Thornton, Elisabeth; McLachlan, Gerry; Sweetman, Dylan; Alberio, Ramiro

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.

Citation

German, S. D., Campbell, K. H., Thornton, E., McLachlan, G., Sweetman, D., & Alberio, R. (2015). Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer. Cellular Reprogramming, 17(1), https://doi.org/10.1089/cell.2014.0071

• Journal Article Type: Article
• Publication Date: Feb 1, 2015
• Deposit Date: Jul 21, 2015
• Publicly Available Date: Jul 21, 2015
• Journal: Cellular Reprogramming
• Print ISSN: 2152-4971
• Electronic ISSN: 2152-4998
• Publisher: Mary Ann Liebert
• Peer Reviewed: Peer Reviewed
• Volume: 17
• Issue: 1
• DOI: https://doi.org/10.1089/cell.2014.0071
• Public URL: https://nottingham-repository.worktribe.com/output/985078
• Publisher URL: http://www.liebertpub.com/overview/cellular-reprogramming/9/
• Additional Information: Final publication is available from Mary Ann Liebert, Inc., publishers http://dx.doi.org/10.1089/cell.2014.0071.