Defining the Integration Capacity of Embryonic Stem Cell-Derived Photoreceptor Precursors

Abstract

Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ESCs with defined factors. In this study, we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ESC-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx 1 and Rhodopsin 1 photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ESC-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ESCderived cells expressed Crx. We conclude that exclusion of proliferative cells from ESC-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ESC-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina. STEM CELLS

Migration, Integration and Maturation of Photoreceptor Precursors Following Transplantation in the Mouse Retina

“…The capacity to migrate may thus be restricted even within the postmitotic precursor population. Defining whether such a window exists is of crucial importance for improving transplantation efficiency, for the identification of accurate cellsurface markers [8,10], and for the isolation of a homogeneous population of integration-competent donor cells from the otherwise heterogeneous populations that arise from stem cell differentiation protocols [11,13,16,58]. Here we found that cells that migrate into the recipient retina are universally rod a-transducin -ve , despite the presence of rod a-transducin + ve cells in the subretinal space for at least 6 weeks posttransplantation (indicating that survival of more mature cells is not an issue).…”

“…These findings have renewed interest in the potential to generate new photoreceptor precursors from stem cell sources [11][12][13]. Ground-breaking studies that utilize 3D culture systems have shown that embryonic stem cells (ESCs) self-organize and mimic the morphological development of the retina [14,15].…”

Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement

“…This has been determined by comparing different protocols for mESC retinal differentiation. Some of the expression levels of photoreceptor markers are lower in adherent two-dimensional (2D) differentiation cultures compared to adult retinas 113,114 ; even though 60% of mESC-derived cells expressed CRX, these cells did not show any mature integrated photoreceptors after transplantation, 114 suggesting they were not transplantation competent. The development of the new generation of 3D retinal differentiation protocols that mimic embryonic morphogenesis of the optic vesicle 52,115 has provided the opportunity to test the importance of developmental stage to enhance transplantation competency.…”

“…52 Importantly, rod photoreceptor precursors obtained from mESC with this protocol were able to integrate and mature in the adult degenerate retina, 116,117 contrary to what had been observed with a 2D retinal differentiation protocol. 114 Using this new advanced 3D protocol, derived rod precursors transplanted in a degenerate adult retina successfully formed inner and outer segments, which were correctly orientated toward the RPE of the host retina. 116 Transplanted cells also formed synapses and were functionally active.…”

“…For example, enhanced photoreceptor yield may be achieved by controlling the size of the EB. 114,124 Formation of a ciliary margin stem cell niche in 3D optic vesicles was recently shown to expand the generation of the retinal progenitor cell population in vitro. 125 Further definition of conditions that expand progenitor populations committed to produce rod or cone photoreceptors would be very useful.…”