43 Induced Pluripotent/Totipotent Stem Cells: Inexhaustible Genetic Stocks for Regeneration and Development of Regionally Adapted Climate-Resilient Livestock

Abstract

Abstract Escalating impacts of climate change present a credible threat to livestock-based food production. Increasing environmental challenges directly affect animal fitness, production performance, and reproductive efficiency with accumulative morbidities and mortality. Albeit extant livestock breeds are adapted to different climate extremes, the steep trajectory of decline in genetic diversity poses a serious threat to the sustenance of livestock farming. After pluripotent stem cells (PSCs) were derived for mice more than 30 years ago, scientists were quick to recognize PSCs as an approach to accelerate livestock biotechnology and assisted reproduction. We posited that core PSC characteristics such as indefinite self-renewal make them ideal for setting up an inexhaustible diploid livestock genetic resource bank. Nevertheless, it has been difficult to derive and sustain naïve PSCs from ruminants. Even reports of generating induced-PSCs (iPSCs) via reprogramming somatic cells that circumvents the embryo and factors such as sex, age or reproductive status, has been crippled with reproducibility issues precluding any livestock applications. In recent systematic studies, we documented two roadblocks: First, that signaling pathways necessary to sustain self-renewal of PSCs from cattle remained unknown. Second, bovine somatic cells have a stable epigenome that is resistant to complete reprogramming to pluripotency. Through systems biology examination of signal homeostasis in the bovine blastocyst inner cell mass (ICM), we uncovered that transforming growth factor (TGF) β effects are actively perturbed in the bovine ICM. This led to the discovery that inhibition of downstream ALK 4/5/7 together with GSK3β and MEK1/2 signaling, supported robust in vitro self-renewal of naïve bovine iPSCs. Simultaneously, we uncovered that epigenetic regulation by the SV40 large T antigen together with the reprogramming genes (OCT4, SOX2, KLF4 and MYC) could result in not only complete reprogramming, but also cells that are set to a stable ground state of pluripotency akin to totipotent stem cells (TPSCs) seen in 16-cell-stage bovine embryos. Cloning performed using the induced TPSCs (iTPSCs) showed significantly greater efficiency of blastocyst formation. Surprisingly, applying identical methods to murine somatic cells also resulted in iTPSCs that can contribute to placenta formation in vivo indicating that the mechanistic basis for sustaining the ground state of developmental plasticity was conserved across species. These findings have now defined a high-fidelity platform for achieving iPSC-based biotechnologies (gene editing and cloning) and for exploring different ruminant livestock applications that have not been previously practicable. Of central importance, these findings also open a path to establishing a live regenerative repository of inexhaustible diploid livestock that can secure the evolving genetic needs for sustaining animal production into the future. Permanent assets of genetic diversity preserved as iTPSCs would ensure rapid resilience in production, health and fertility through propagating heterosis and enabling genetic fixes for regional climate-resilient livestock phenotypes that better tolerate environmental extremes.