Abstract
Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by ectopic expression of transcription factors or small molecule treatment, which resemble embryonic stem cells (ESCs). They hold great promise for improving the generation of genetically modified large animals. However, few porcine iPSCs (piPSCs) lines obtained currently can support development of cloned embryos. Here, we generated iPSCs from porcine adipose-derived stem cells (pADSCs), using drug-inducible expression of defined human factors (Oct4, Sox2, c-Myc and Klf4). Reprogramming of iPSCs from pADSCs was more efficient than from fibroblasts, regardless of using feeder-independent or feeder-dependent manners. By addition of Lif-2i medium containing mouse Lif, CHIR99021 and PD0325901 (Lif-2i), naïve-like piPSCs were obtained under feeder-independent and serum-free conditions. These successfully reprogrammed piPSCs were characterized by short cell cycle intervals, alkaline phosphatase (AP) staining, expression of Oct4, Sox2, Nanog, SSEA3 and SSEA4, and normal karyotypes. The resemblance of piPSCs to naïve ESCs was confirmed by their packed dome morphology, growth after single-cell dissociation, Lif-dependency, up-regulation of Stella and Eras, low expression levels of TRA-1-60, TRA-1-81 and MHC I and activation of both X chromosomes. Full reprogramming of naïve-like piPSCs was evaluated by the significant up-regulation of Lin28, Esrrb, Utf1 and Dppa5, differentiating into cell types of all three germ layers in vitro and in vivo. Furthermore, nuclear transfer embryos from naïve-like piPSCs could develop to blastocysts with improved quality. Thus, we provided an efficient protocol for generating naïve-like piPSCs from pADSCs in a feeder-independent and serum-free system with controlled regulation of exogenous genes, which may facilitate optimization of culture media and the production of transgenic pigs.
Highlights
Embryonic stem cells (ESCs) are highly promising in regenerative medicine for their potential to differentiate into cells from all three embryonic germ layers
Characterization of porcine adipose-derived stem cells (pADSCs) Approximately 30–50 ml of porcine subcutaneous adipose tissue was obtained from one piglet
Porcine ADSCs isolated from the tissue showed a spindle-like fibroblastic morphology (Fig.1A), and could proliferate rapidly in low passage (Fig S1), which managing to undergo 20 passages without significant replication senescence
Summary
Embryonic stem cells (ESCs) are highly promising in regenerative medicine for their potential to differentiate into cells from all three embryonic germ layers. Since iPSCs, which share similar properties to ESCs [2], have been successfully derived from somatic cells with ectopic expression of transcription factors Oct, Sox, c-Myc and Klf4 [3], a new type of cells that avoids the abovementioned hurdles was provided. This may offer greater potential for in vitro disease modeling, drug screening and regenerative cell therapy. As the limited proliferative capacity and low frequency of homologous recombination of somatic cells, ESCs are regarded as a powerful cell resource for generating genetically modified animals, which can serve to increase our knowledge of mammalian physiology and disease [4]. Difficulties remain in generating healthy offspring from piPSCs [4], and the quality of piPSCs may be the key factor affecting success of this aim
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