Generation of Induced Pluripotent Stem Cells in Rabbits

Abstract

Human induced pluripotent stem (iPS) cells have the potential to establish a new field of promising regenerative medicine. Therefore, the safety and the efficiency of iPS-derived cells must be tested rigorously using appropriate animal models before human trials can commence. Here, we report the establishment of rabbit iPS cells as the first human-type iPS cells generated from a small laboratory animal species. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2, and OCT3/4) were introduced successfully into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells; they formed flattened colonies with sharp edges and proliferated indefinitely in the presence of basic FGF. They expressed the endogenous pluripotency markers c-MYC, KLF4, SOX2, OCT3/4, and NANOG, whereas the introduced human genes were completely silenced. Using in vitro differentiating conditions, rabbit iPS cells readily differentiated into ectoderm, mesoderm, and endoderm. They also formed teratomas containing a variety of tissues of all three germ layers in immunodeficient mice. Thus, the rabbit iPS cells fulfilled all of the requirements for the acquisition of the fully reprogrammed state, showing high similarity to their embryonic stem cell counterparts we generated recently. However, their global gene expression analysis revealed a slight but rigid difference between these two types of rabbit pluripotent stem cells. The rabbit model should enable us to compare iPS cells and embryonic stem cells under the same standardized conditions in evaluating their ultimate feasibility for pluripotent cell-based regenerative medicine in humans.

Rabbit as a reproductive model for human health

“…Moreover, ES cell isolation has been reported (Honda et al 2008, Intawicha et al 2009, while the production of chimera using these cells remains rare (Zakhartchenko et al 2011) and no germinal chimera have been reported yet. However, isolation of iPS cells should help the development of functional genomics analyses in this species (Honda et al 2010). Finally, the concomitant development of genome data together with new transgenesis strategies (Duranthon et al 2012), notably the possibility to target specific genes by injection of zinc finger nucleases into embryo cytoplasm , should promote the rabbit to its legitimate place among the standard models for developmental biology and reproductive pathologies.…”

Germ cell specification and pluripotency in mammals: a perspective from early embryogenesis

“…EGCs are established from mouse PGCs [8, 9] and have been attempted from human [46], rabbit [87], pig [84] and cattle [84] PGCs. After the discovery of iPSCs from mouse cells [88], this was followed by human [89], monkey [90], rabbit [91], and pig [84]. …”

“…LIF and its related pathways are dispensable for maintenance of undifferentiated status in primate, rabbit, pig ESCs [103, 104]. Treatment of rabbit ESCs with Rho-associated kinase (ROCK) inhibitor, Y27632, significantly enhanced cell growth similar to human ESCs [91, 105, 106]. Although there is little effect of FGF2 addition on the growth of monkey ESCs [107], FGF2 and Activin/Nodal signaling can maintain the undifferentiated status through Smad2/3 activation of rabbit and porcine ESCs and iPSCs [103, 105].…”

“…Canine PSCs express SSEA1, SSEA4, TRA1-60, TRA1-81, and Rex1 [113]. In rabbit ESCs, SSEA-1, SSEA-3, SSEA-4, TRA-1–60 and TRA-1–81 are not detectable [81, 82, 91]. …”

Induced pluripotent stem cells: opportunities and challenges

“…Twenty-four candidate factors were transduced into mouse embryonic fibroblasts (MEFs) by retroviral delivery and this identified four factors that can convert fibroblasts into induced pluripotent stem (iPS) cells [6]. iPS cells have been generated from mouse [6], rat [7,8], monkey [9], pig [10], dog [11], rabbit [12] and human [13,14]. Most of the iPS cells are derived using the Oct3/4, Sox2, Klf4 and c-Myc reprogramming factors.…”