Generation of Pig iPS Cells: A Model for Cell Therapy
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Reprogramming of pig somatic cells to induced pluripotent stem cells provides a tremendous advance in the field of regenerative medicine since the pig represents an ideal large animal model for the preclinical testing of emerging cell therapies. However, the current generation of pig-induced pluripotent stem cells (piPSCs) require the use of time-consuming and laborious retroviral or lentiviral transduction approaches, in order to ectopically express the pluripotency-associated transcription factors Oct4, Sox2, Klf4 and c-Myc, in the presence of feeder cells. Here, we describe a simple method to produce piPSC with a single transfection of a CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc and a green fluorescent protein (GFP) reporter gene, in gelatine-coated plates, with or without feeder cells. In our system, the derivation of piPSCs from adult pig ear fibroblasts on a gelatine coating showed a higher efficiency and rate of reprogramming when compared with three consecutive retroviral transductions of a similar polycistronic construct. Our piPSCs expressed the classical embryonic stem cell markers, exhibit a stable karyotype and formed teratomas. Moreover, we also developed a simple method to generate in vitro spontaneous beating cardiomiocyte-like cells from piPSCs. Overall, our preliminary results set the bases for the massive production of xeno-free and integration-free piPSCs and provide a powerful tool for the preclinical application of iPSC technology in a large animal setting.
KeywordsiPS cells Embryonic stem cells Clinical translation Cardiomyocyte differentition
We are grateful to Meritxell Carrió and Laetitia Casano for expert assistance with cell culture techniques, José Miguel Andrés Vaquero for assistance with flow cytometry, Lola Mulero Pérez, Cristina Pardo and Mercé Gaudes Martí for bioimaging assistance, Cristina Gómez and Cristina Morera for expert assistance in molecular biology techniques. NM was partially supported by Juan de la Cierva Program, EG was partially supported by Sara Borrell Program, AMCR supported by Fundação para a Ciência e Tecnologia. This work was partially supported by grants from MICINN, TERCEL, CIBER and Fundación Cellex.
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