Abstract
Purpose::
To characterize a population of late, glial-restricted human prenatal retinal progenitors (hRPC) and to determine if mis-expression of Mash1, a transcription factor crucial for neurogenesis, can reprogram them to adopt a neuronal cell fate.
Methods::
hRPC were isolated from prenatal human retina (54-120 days post-conception) and cultured as neurospheres for up to one year in defined medium supplemented with EGF, FGF2 and conditioned media (CM) from parallel human prenatal retinal pigmented epithelium (RPE) cultures. Proliferating and differentiated hRPC at early and late passage were examined by immunocytochemistry, immunoblot and PCR analysis to determine protein and RNA expression profiles. For Mash1 mis-expression, long-term hRPC neurosphere cultures were plated as monolayers and infected for 24 hr with a lentiviral Mash1 construct (250 ng p24/106 cells). Infected cells were maintained for 24 hr in RPE CM, differentiated in the absence of mitogens for an additional 5 days and examined by light microscopy and immunocytochemistry.
Results::
Virtually all hRPC expressed neural stem cell proteins such as nestin (91.5 ± 4.2%), sox2 (92.9 ± 2.5%), and vimentin (94.6 ± 3.7%), as well as many neural and eye specification genes including Pax6, Crx, Rax, Chx10 and Hes1. The expression of Flk1, BLBP, and Glast1 indicated that hRPC possess a radial glia-like character. Upon differentiation at early passage, these cultures produced neurons and glia, but at later passages (>12 weeks) became exclusively gliogenic. However, when hRPC were treated with a lentiviral Mash1 construct and differentiated, 25.8% of the infected cells were ß-III tubulin immunopositive and had long, bipolar processes.
Conclusions::
These findings show that hRPC exhibit a progressive change in cell fate potential over time in culture but can demonstrate competency for neurogenesis by manipulating their intrinsic milieu.
Keywords: retinal development • differentiation • retinal culture