Isolation and Expansion of Adult Human Eye Neural Progenitors

Abstract

Abstract: : Purpose: Isolate and generate high amounts of adult human retinal progenitors. Method: After full retinal development, retinal stem cells can be retrieved in adult mice at the level of the ciliary margin. By anatomical homology, we isolated pigmented cells from the pars plana and the pars plicata of human donors of various ages using enzymatic dissociation. Cells were grown in DMEM/F12 medium and N1 supplement. Results: A subpopulation of pigmented cells proliferates in response to insulin only, forming big clusters of cells termed spheres. The presence of EGF and FGF-2 increased the number of proliferating progenitors. No differences in sphere growth or number were observed between the donors, including for people aged 80, showing a remarkable conservation of this pool of cells throughout life. The kinetics of sphere growth is comparable to that observed in mice. After cell differentiation induction, single spheres generated both neuron-like and glia cells attested by the expression of b-tubulin-III and vimentin respectively as well as by their morphology. Expansion of neural progenitors can be achieved by controlled conditions: a 10 million-fold expansion can be reached in 42 days. The enzymatic composition used for primary cell dissociation is crucial for the success of cell expansion. After expansion, cells maintain the ability to generate neurons and glia, but to a lesser extent. Conclusion: It appears that adult human retinal progenitors provide a new tool to study human neurogenesis. Importantly, because these cells are the reminiscent origin of retinal stem cells, they may have the potential to be a source of photoreceptors that can be used for transplantation studies in animal models of retinal degeneration.

Keywords: retinal development • transplantation • retinal pigment epithelium