A defined subset of clonal retinal stem cell spheres is biased to RPE differentiation.

Retinal stem cells (RSCs) are rare pigmented cells found in the pigmented ciliary layer of the mammalian retina. Studies show that RSCs can replicate to maintain the stem cell pool and produce retinal progenitors that differentiate into all retinal cell types. We classified RSCs based on their level and distribution of pigment into heavily pigmented (HP), lightly pigmented (LP), and centrally pigmented (CP) spheres. We report that CP spheres are capable of generating large cobblestone lawns of retinal pigment epithelial (RPE) cells. The other clonal sphere types (HP and LP) primarily produce cells with neural morphology and fewer RPE cells. The RSCs are homogeneous, but their downstream progenitors are different. We found that CP spheres contain highly proliferative populations of early RPE progenitors that respond to proliferative signals from the surrounding non-pigmented cells. HP and LP spheres contain late RPE progenitors which are not affected by proliferative signals.

Expansion of retinal stem cells and their progeny using cell microcarriers in a bioreactor.

Blindness as a consequence of degenerative eye diseases (e.g., age-related macular degeneration and retinitis pigmentosa) is a major health problem and numbers are expected to increase by up to 50% by 2020. Unfortunately, adult mouse and human retinal stem cells (RSCs), unlike fish and amphibians, are quiescent in vivo and do not regenerate following disease or injury. To replace lost cells, we used microcarriers (MCs) in a suspension stirring bioreactor to help achieve numbers suitable for differentiation and transplantation. We achieved a significant 10-fold enrichment of RSC yield compared to conventional static culture techniques using a combination of FACTIII MCs and relative hypoxia (5%) inside the bioreactor. We found that hypoxia (5% O2 ) was associated with better RSC expansion across all platforms; and this can be attributed to hypoxia-induced increases in survival and/or symmetric division of stem cells. In the future, we will target the differentiation of RSCs and their progeny toward rod and cone photoreceptor phenotypes using FACTIII MCs inside bioreactors to expand their populations in order to produce the large numbers of cells needed for transplantation.

Induction of rod versus cone photoreceptor-specific progenitors from retinal precursor cells.

During development, multipotent progenitors undergo temporally-restricted differentiation into post-mitotic retinal cells; however, the mechanisms of progenitor division that occurs during retinogenesis remain controversial. Using clonal analyses (lineage tracing and single cell cultures), we identify rod versus cone lineage-specific progenitors derived from both adult retinal stem cells and embryonic neural retinal precursors. Taurine and retinoic acid are shown to act in an instructive and lineage-restricted manner early in the progenitor lineage hierarchy to produce rod-restricted progenitors from stem cell progeny. We also identify an instructive, but lineage-independent, mechanism for the specification of cone-restricted progenitors through the suppression of multiple differentiation signaling pathways. These data indicate that exogenous signals play critical roles in directing lineage decisions and resulting in fate-restricted rod or cone photoreceptor progenitors in culture. Additional factors may be involved in governing photoreceptor fates in vivo.