Directed Neuronal Specification of Porcine Induced Pluripotent Stem Cells.

Abstract

A recent goal of this laboratory has been to create porcine pluripotent stem cells, i.e. ones capable of differentiating into all cell types of the body thereby expanding the potential use of swine as a biomedical model for studying human disease as well as cells that might be easily modified by gene knock-in/knock-out technologies. Importantly, pig is a potentially useful model for studying human pathologies due to similarities in organ size, immunology and whole animal physiology between the two species. If the safety and efficacy of stem cell transplantation is to be tested in an animal model before being applied to humans, the pig would likely be a species of choice. The ability to derive porcine (p) iPSC lines from a particular outbred animal and conduct tissue transplantation on the same pig later and follow the success of the transplant over the course of months or even years would be a particularly valuable advance. We have recently created piPSC from embryonic fibroblasts and primary umbilical cord mesenchyme by a similar strategy used for the mouse and human, namely ectopically expressing reprogramming genes (OCT4/SOX2/MYC/KLF4) in somatic cells. The piPSC phenotypically resemble human embryonic stem cells (ESC), express the typical gene and surface antigen markers of ESC, proliferate continuously in culture, possess high telomerase activity, form embryoid bodies (EB) and teratomas, where they are able to differentiate along the three main germ line lineages. The present aim has been to demonstrate that piPSC can be directed to differentiate towards neuronal endpoints by using protocols based on those used successfully with human ESC. The piPSC, ID6, grown on a MEF feeder were mechanically detached and formed EB were cultured in ES cell medium containing knockout serum replacement but lacking FGF2 for 4 days. The EB were cultured following two days with serum free medium supplemented 0.1 mg/ml transferrin, 5 µg/ml insulin, 20 nM progesterone, 0.1 mM puterescine, 30 nM sodium selenite and 2 µg/ml heparin. The EB were then collected in the same medium and allowed to settle on dishes plated to which they attached and formed a flattened cell mass within 48 h. The cells differentiated into typical neuronal rosettes by d 9 in the same medium, which had a radially-organized columnar morphology and expressed transcripts for the neuroectodermal transcription factors, PAX6 and OTX2. These experiments demonstrate that the porcine iPSC line, ID6, is capable of being directed along a neuronal lineage by cues that work successfully on human ESC and iPSC, raising the possibility to achieve controlled differentiation of this same kind of cell along additional lineages. They provide confidence that porcine iPSC may soon be employed to provide tissue grafts that can be matched genetically to recipients and tested for their safety in transplantation. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD21896. (poster)