Derivation of photoreceptor precursors from human Müller stem cells and their application in experimental photoreceptor replacement

The adult human retina has a population of Müller glia that exhibit stem cell characteristics. The study investigated the feasibility of obtaining Müller stem cells from small samples of human, rat, rabbit and nonhuman primate retinae in order to provide proof of concept for the development of patient specific therapies. It also explored the role of human Müller stem cells in providing a source of rod photoreceptor precursors that may be applied to experimental photoreceptor replacement. Müller glia with stem cell characteristics were readily obtained from small samples of fresh cadaveric human retina and retinae from rats and non-human primates. However derivation of cells from human retinectomy specimens was not possible, with the marked inflammatory environment likely to be inhibiting proliferation of these cells in vitro. As determined by quantitative PCR and immunocytochemistry, in vitro culture of Müller stem cells in the presence of FGF2, taurine, retinoic acid and IGF-1 led to increased expression of the photoreceptor markers CRX, NR2E3 and rhodopsin, associated with increased gene expression of components of the phototransduction cascade. The expression of NRL was not modified, suggesting that the differentiation of adult human Müller stem cells towards photoreceptors may occur through a pathway independent of this transcription factor. In vitro functional analysis suggested an increased responsiveness of differentiated cells to cGMP analogues and light stimulation as judged by calcium imaging and patch clamp techniques. Following subretinal transplantation into rodent models of photoreceptor degeneration, Müller stem cell derived photoreceptor precursors migrated and integrated into the outer nuclear of degenerate rodent retina, demonstrating expression of photoreceptor markers and local synapse formation. Grafting of photoreceptor differentiated but not undifferentiated cells led to a significant increase in rod photoreceptor function as shown by the scotopic electroretinogram (ERG). The present observations show that human Müller stem cells have the capacity to differentiate into photoreceptor precursors and that these have the potential to be used in the development of therapies for human photoreceptor disease