Differentiation of Swine iPSC into Rod Photoreceptors and Their Integration into the Retina

• Published in: Stem cells (Dayton, Ohio) Ohio), 2011-06, Vol.29 (6), p.972-980
• Main Authors: Zhou, Liang, Wang, Wei, Liu, Yongqing, de Castro, Juan Fernandez, Ezashi, Toshihiko, Telugu, Bhanu Prakash V.L., Roberts, R. Michael, Kaplan, Henry J., Dean, Douglas C.
• Format: Article
• Language: English
• ISSN: 1066-5099; 1549-4918
• DOI: 10.1002/stem.637

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments utilizing stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with swine induced pluripotent stem cells (iPSC). Here, we subjected swine iPSC to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1 , NANOG and SOX2 and induction of rod photoreceptor genes RCVRN , NRL , RHO and ROM1 . While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of RHO and ROM1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO + cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that swine iPSC can differentiate into photoreceptors in culture and these cells can integrate into the damaged swine neural retina thus laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.