Abstract
Purpose: :
Recently, our laboratory has shown that the genes NeuroD1 and NeuroD4 have a similar expression profile to NRL in the developing mouse retina, and may thus play a role in photoreceptor differentiation and integration. To determine if the transient expression of such key transcription factors could induce a photoreceptor phenotype in pluripotent cells or induce transdifferentiation in human RPE in vitro, the genes NRL, CRX, NeuroD1 and NeuroD4 were forcibly expressed using ex vivo gene therapy in the immortalized cell line ARPE-19, and in stem cell and neural progenitor cell lines.
Methods: :
ARPE-19, human embryonic stem cells (hESC) and neural progenitors (stem cells cultured on PA6 cells x14 days) were each transfected with expression constructs containing the genes NRL, CRX, NeuroD1 and NeuroD4 individually and in combination. Cells were then incubated for 72 hours, then fixed and examined using immune cytochemical staining, and studied using western blot and PCR for markers of the photoreceptor phenotype. Total RNA was extracted from transfected cells, and their gene expression profiles were studied using microarray with the Affymetrix human genome chip U133 PLUS 2.0.
Results: :
Immune cytochemistry showed positive staining of RPE and hESC transfected with both NRL and NeuroD1 for several photoreceptor markers, including recoverin and rhodopsin. These findings were confirmed using PCR and western blot. Microarray data showed a global shift of gene expression towards a neural retina profile, including significant expression of recoverin and rhodopsin.
Conclusions: :
RPE represents an autologous source of precursor cells that are readily attainable, and that can be differentiated into photoreceptor phenotype by plasmid expression of NRL and NeuroD1.
Keywords: retinal pigment epithelium • gene transfer/gene therapy • retinal degenerations: cell biology