Reprogramming chick RPE progeny cells to differentiate towards retinal neurons by ash1.
OBJECTIVE
Harnessing a cell culture of retinal pigment epithelium (RPE) to give rise to retinal neurons may offer a source of developing neurons for cell-replacement studies. This study explores the possibility of reprogramming RPE progeny cells to differentiate toward retinal neurons with achaete-scute homolog 1 (ash1), a proneural gene that is expressed in progenitor cells in the developing retina and promotes amacrine cell production when overexpressed in the chick retina.


METHODS
Replication Competent Avian Splice (RCAS) retrovirus was used to drive the ectopic expression of ash1 in cell cultures of dissociated RPE isolated from day 6 chick embryos. RCAS expressing green fluorescent protein (RCAS-GFP) was used as control. The cultures were examined for de novo generation of neuron-like cells by molecular, cellular, and physiologic criteria.


RESULTS
In control cultures infected with RCAS-GFP, RPE cells appeared cobblestone-like and often darkly pigmented. In cultures infected with RCAS-ash1, however, cells remained de-pigmented and frequently formed clusters. Further examination at the morphological and molecular levels showed the development of elaborate processes characteristic of neurons and the expression of genes/markers that identify different types of retinal neurons. The most prevalently expressed neural marker was calretinin, which in the chick retina identifies amacrine, ganglion, and horizontal cells. As an assay for functional maturation, the reprogrammed cells were analyzed for the presence of functional, ionotropic glutamate receptors that lead to a rise in the cytosolic free calcium (Ca(2+)) concentration. Calcium imaging showed that reprogrammed cells responded to glutamate and N-methyl-D-aspartate (NMDA) by increasing their Ca(2+) concentrations, which, after reaching a peak level, returned to the basal level. The response curves of reprogrammed cells resembled those of cultured retinal neurons.


CONCLUSIONS
These results suggest that RPE progeny cells can be reprogrammed by ash1 to develop molecular, morphological, and physiologic properties that are characteristic of retinal neurons.
Mao
Weiming
W
Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, AL, USA.
Yan
Run-Tao
RT
Wang
Shu-Zhen
SZ
D000818:Animals; D030161:Avian Proteins / Q000235:genetics / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000187:drug effects; D002642:Chick Embryo; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D007150:Immunohistochemistry; D016202:N-Methylaspartate / Q000494:pharmacology; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055351:Retinal Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D012190:Retroviridae / Q000502:physiology; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D030161:Avian Proteins; D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D018698:Glutamic Acid; D005998:Glycine; D016202:N-Methylaspartate; D014746:Vimentin; C099107:CASH-1 protein, Gallus gallus