Development of defined culture condition for pig pluripotent stem cells and their potential application

Abstract

Derivation of pluripotent cells can be accomplished by in vitro-culture of early embryos. Pluripotent stem cells (PSCs) have been considered as a candidate for regenerative medicine and cell therapy. PSC lines derived from domestic animals such as pigs and cattle are useful tools in the production of transgenic animals. Especially, because of the physiological and immunological similarities between pigs and humans, porcine PSCs have been identified as a useful candidate for a certain human disease. So, in this study, pig PSCs were derived from various origins including embryos and somatic cells to find their application. Firstly, I tried to analyze stem cells derived from embryos and fetus for unveiling mechanism of pluripotency in pig. So, pig embryonic stem cells (ESCs) were derived from in vitro-produced embryos by supplementing FGF2. And reprogramming of PGCs and maintenance of EGCs were achieved by FGF2 signaling. The results showed that FGF2 signaling has a pivotal roles in establishing and maintaining pluripotency in pig both PSCs. Next, pig somatic cells were reprogrammed into pluripotent state using Yamanakas factors. During reprogramming, FGF2 treatment strongly up-regulated specific pluripotent genes such as SOX2, KLF4, REX1, and epithelial-specific markers when compared to LIF treatment, and blocking FGF2 signaling down-regulated KLF4 and NANOG. Then, optimization of culture media for pig ESCs were conducted by using various metabolic components and signaling molecules. As a result, pig ESCs were successfully established by chemically defined media supplemented with FGF2, ACTIVIN A and WNT activator. These cells expressed pluripotent genes such as OCT4, SOX2 and NANOG, and could be maintained for extended periods. Next, transgenic pluripotent cell lines were generated by lentiviral vector harboring enhanced green fluorescence protein (EGFP). Transgenes were successfully introduced into ESCs and transfection was the most efficient under multiplicities of infection (MOI) of 75. It was apparent that the expression of inserted lentiviral transgenes was controlled by DNA methylation. Neuronal progenitor cells were derived from pig embryonic germ cells. Similar with other species, neuronal progenitor cells were successfully induced by treatment of retinoic acid and these cells expressed neuronal markers such as PAX6, NESTIN and SOX1. Taken together, I found that, as a non-permissive species, pig PSCs are maintained by mainly FGF signaling, and additional signaling molecules such as ACTIVIN and WNT are required for supporting pluripotency. And pig ESCs could be derived using chemically defined media supplementing FGF2, ACTIVIN A and WNT. This study will not only provide basic understanding for mechanism of maintaining pluripotency but also apply stem cell engineering for regenerative medicine. Accordingly, studies on pig PSCs will pave the way for human cell therapy and shed new light on researches of PSCs.