Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig

Abstract

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model. Induced nuclear reprogramming through induced pluripotent stem cell (iPS)2 technology is an amazing achievement full of challenge to the intellect and important practical implications (1, 2). Overexpression of exogenous factors that are highly enriched in embryonic stem cell (ESC) can rearrange the genetic program of different cell types, including somatic and adult stem cells, and induce a long lasting ESC-like pluripotent state (3-7). The repercussions of iPS technology are vast: it provides a way to create patient-specific stem cells that bypasses ethical and technical issues surrounding human ESC derivation and somatic cell nuclear transfer (8, 9), a state of the art model for studying genetic diseases in vitro (10, 11), and an incredible backwards route that can crystallize our current understanding of developmental and stem cell biology. Many questions, especially mechanistic, remain unanswered, but the current rhythm of research may bring iPS to clinical application sooner than expected. However, before jumping onto such extraordinary endeavor, safety must be scrupulously tested in an animal model close enough to humans. Nowadays that iPS technology is expanding, with improved delivery systems, chemical additions, new tissue culture conditions, and multiple cell sources being reported regularly, such animal model is essential to set up quality standards (12-18). Mice, and maybe rats, will possibly continue unrivalled as the easier ways to learn about reprogramming machinery and improve methodology, but their size, physiology, and reduced lifespan are handicaps for making serious assumptions regarding safety in humans. Given philogenetic similarity, monkeys are theoretically an excellent alternative, but in practice ethical concerns remain to at least some extent, and they are neither easy to maintain nor to breed. Swine, a regular source of food whose farming humans have adapted over myriads of years and whose physiology is r...

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

“…Gene targeting by homologous recombination is extremely inefficient in porcine somatic cells 3 , and porcine embryonic stem cells (ES) have not yet been established 15 . Recently, several groups described the derivation of porcine induced pluripotent cells (iPS) [16][17][18][19][20] , however, only low chimerism has been found after blastocyst complementation experiments 21,22 . The employment of porcine iPS cells in the SCNT method resulted in extremely low rates of born piglets 23 .…”

Overcoming barriers to the clinical utilization of iPSCs: reprogramming efficiency, safety and quality

“…Through iterative experiments, they eventually found that all factors save four were dispensable for reprogramming-these four factors became known as the classical quartet of pluripotential reprogramming factors; Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006). This classical cocktail of reprogramming factors has been shown to induce pluripotency within a multitude of mouse cell types, as well as rhesus monkey (Li et al, 2009b;Liao et al, 2009), pig (Esteban et al, 2009;Ezashi et al, 2009;Wu et al, 2009;West et al, 2010) and human cells (Takahashi et al, 2007;Yamanaka, 2007).…”

“…Despite this, pluripotent stem cell lines for the rat, pig and monkey in the form of iPSC lines have been derived through the overexpression of Oct4, Sox2, Klf4, and c-Myc from these species Esteban et al, 2009;Ezashi et al, 2009;Li et al, 2009b;Liao et al, 2009;Wu et al, 2009). However, we note that some caution must be taken when considering these iPSC lines from such species, because the pluripotential character of such species' iPSC lines is not comparable with the pluripotent character of conventional mouse ES and iPSC cell lines.…”

“…However, we note that some caution must be taken when considering these iPSC lines from such species, because the pluripotential character of such species' iPSC lines is not comparable with the pluripotent character of conventional mouse ES and iPSC cell lines. For example, pig iPSCs do not exhibit as robust reactivation of endogenous pluripotency genes as mouse iPSCs, and they are incapable of silencing integrated genes (Esteban et al, 2009;Ezashi et al, 2009;Wu et al, 2009). Nevertheless, porcine iPSCs derived from mesenchymal cells are capable of contribution to all three germ layers after injection into the early embryo, affirming that they at least are capable of multilineage differentiation (West et al, 2010).…”

“…Tbx3 is necessary for safeguarding the pluripotency of mouse ES cells, as its knockdown elicits © Higher Education Press and Springer-Verlag Berlin Heidelberg 2012 differentiation (Ivanova et al, 2006). It appears to have pleiotrophic roles, as it is also required for mammary bud development and limb development (Carlson et al, 2002;Esteban et al, 2009;Graf et al, 2009;Maekawa et al, 2011). Furthermore, overexpression of Tbx3 in conjunction with Myc or oncogenic Ras leads to the efficient oncogenic transformation of mouse fibroblasts; it appears that Tbx3's role in oncogenic cooperation is to inhibit the induction of p53 and p19 ARF that is typically triggered by Myc and Ras (Carlson et al, 2002).…”