Establishment of induced pluripotent stem cells from pig adipose tissue-derived fibroblasts
Objectives: Pluripotent stem cells are one of the most promising cell sources for cardiac regenerative therapy. Nevertheless, currently no large animal models are available for adequate preclinical evaluation of feasibility, efficiency and safety. Therefore, we focused on the generation of porcine induced pluripotent stem cells (iPS).
Methods: Adult fibroblasts derived from porcine adipose-tissue were reprogrammed to iPS by infection with the lentiviral vector phuSTEMCCA which constitutively expresses the four Yamanaka factors. Expression of transgenes and corresponding endogenous counterparts was demonstrated using species-specific primers. Further porcine pluripotency markers were detected by qRT-PCR or fluorescent reporter probes (SmartFlares™, Merck Millipore) in live cells. Upon differentiation gene expression specific for the three different germ layers was evaluated by qRT-PCR.
Results: Approximately two weeks after transduction first iPS-like colonies appeared which showed a flattened morphology similar to established human iPS cells. The porcine iPS cells strongly expressed the four human transgenes (OCT4, KLF4, SOX2 and C-MYC) and the mRNA of all four endogenous counterparts could be detected by qRT-PCR. In addition, established iPS clones also expressed the pluripotency markers REX1, NANOG and GDF3. The expression of mRNA for NANOG and GDF3 was further confirmed by live staining with SmartFlares™. Established porcine iPS colonies could be cultured over more than 50 passages. Upon differentiation, endoderm markers AFP and FOXA2, absent in undifferentiated iPS cells, became detectable. Mesoderm-specific expression of HAND1 was strongly upregulated (more than 200-fold) while ectodermal reporter genes showed a rather modest increase.
Conclusion: Our results support the successful reprogramming of somatic fibroblasts into different porcine iPS lines as a first step towards a large animal model for cardiac regeneration. Although the in-vitro differentiation is still insufficient in porcine iPS lines the expression of all four endogenous reprogramming genes and further pluripotent marker genes as well as the stable growth in culture confirm the pluripotent state of generated iPS lines.