Abstract
Purpose: :
Outer retinal degeneration with photoreceptor (PR) loss is the cause of many incurable forms of blindness. Cell transplantation remains an important option, particularly in cases where a substantial loss of PRs has already occurred. Recent successes in the field have identified some of the key chemical and biological cues which drive PR derivation; however the efficiency of this process remains limited and usually requires extensive periods in vitro.
Methods: :
We have developed a differentiation regime which rapidly and efficiently derives highly enriched populations of PR precursors from human embryonic stem cells (hESC). This method is serum and feeder free and utilises a three-step protocol designed to mimic the events leading to PR genesis during normal retinal development incorporating 1) anterior neural specification, 2) retinal determination and 3) PR specification and differentiation.
Results: :
We report that hESC differentiating under our regime over 60 days sequentially acquire markers associated with neural, retinal field and PR cells. By qPCR, flow cytometry and immunocytochemical analysis we demonstrate robust upregulation of neuroectodermal markers NCAM1 (neural cell adhesion molecule 1) and NEUROD (neurogenic differentiation 1) by 30 days, eye development transcription factor PAX6 (paired box 6), retinal progenitor and PR precursor markers CHX10 (ceh-10 homeo domain containing homolog) and CRX (cone-rod homeobox) over days 30 to 45 and PR-specific markers RCVRN (recoverin), OPN1SW (opsin 1), Rhodopsin and ARR3 (arrestin 3) over days 45 to 60. In an adapted version of our protocol we also demonstrate the efficient production of retinal pigmented epithelium (RPE), an additional important replacement cell type for retinal stem cell therapy.
Conclusions: :
The development of more efficient methods by which to derive de novo PRs for cell replacement in outer retinal degeneration has clear implications. Our differentiation regime robustly and efficiently yields expandable populations of enriched PR or RPE precursors from hESC with higher efficiency and in a shorter time frame than what has been published previously.
Keywords: photoreceptors • degenerations/dystrophies