TY - JOUR
T1 - Generation of Light-Sensitive Photoreceptor Phenotypes by Genetic Modification of Human Adult Ocular Stem Cells with Crx
AU - Jomary, Catherine
AU - Jones, Stephen E.
AU - Lotery, Andrew J.
PY - 2010/2
Y1 - 2010/2
N2 - PURPOSE. This study compared the effect of the transcription factor Crx (cone, rod homeobox) on the differentiation of human adult corneal (hCSC) and retinal (hRSC) stem cells into functional photoreceptors.
METHODS. Stem cells isolated from postmortem human corneas and retinal ciliary bodies were maintained in serum-free culture and genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Expression of stem cell markers (Pax6, Oct3/4, and proliferating cell nuclear antigen [PCNA]), neuronal markers (nestin, neuron-specific class III beta-tubulin, Map2 a/b, and neurofilament), and photoreceptor-specific markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, and beta-6-PDE) was evaluated by immunocytochemistry. A cGMP enzyme-linked immunoassay was used to assess phototransduction cascade activity by measurement of light-induced hydrolysis of cGMP.
RESULTS. Expression of the stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4 was decreased by exogenous Crx expression in both hCSCs and hRSCs. Correspondingly, the expression of the mature neuronal markers Map2 a/b and neurofilament was increased. Both hCSCs and hRSCs displayed photoreceptor-specific immunolabeling. However, light-activated GMP hydrolysis was observed only in hRSCs after exogenous expression of Crx.
CONCLUSIONS. The present study extends previous findings that exogenous Crx expression can promote differentiation of human retina-derived stem cells into light-sensitive photoreceptor phenotypes. Although Crx can induce human cornea-derived stem cells to express photoreceptor-specific proteins, it does not seem to be sufficient to direct their differentiation into functional photoreceptors. Nevertheless, this study demonstrates that genetic modification of adult human retinal stem cells can cause differentiation into light-sensitive photoreceptor phenotypes. (Invest Ophthalmol Vis Sci. 2010; 51: 1181-1189) DOI: 10.1167/iovs.09-4385
AB - PURPOSE. This study compared the effect of the transcription factor Crx (cone, rod homeobox) on the differentiation of human adult corneal (hCSC) and retinal (hRSC) stem cells into functional photoreceptors.
METHODS. Stem cells isolated from postmortem human corneas and retinal ciliary bodies were maintained in serum-free culture and genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Expression of stem cell markers (Pax6, Oct3/4, and proliferating cell nuclear antigen [PCNA]), neuronal markers (nestin, neuron-specific class III beta-tubulin, Map2 a/b, and neurofilament), and photoreceptor-specific markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, and beta-6-PDE) was evaluated by immunocytochemistry. A cGMP enzyme-linked immunoassay was used to assess phototransduction cascade activity by measurement of light-induced hydrolysis of cGMP.
RESULTS. Expression of the stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4 was decreased by exogenous Crx expression in both hCSCs and hRSCs. Correspondingly, the expression of the mature neuronal markers Map2 a/b and neurofilament was increased. Both hCSCs and hRSCs displayed photoreceptor-specific immunolabeling. However, light-activated GMP hydrolysis was observed only in hRSCs after exogenous expression of Crx.
CONCLUSIONS. The present study extends previous findings that exogenous Crx expression can promote differentiation of human retina-derived stem cells into light-sensitive photoreceptor phenotypes. Although Crx can induce human cornea-derived stem cells to express photoreceptor-specific proteins, it does not seem to be sufficient to direct their differentiation into functional photoreceptors. Nevertheless, this study demonstrates that genetic modification of adult human retinal stem cells can cause differentiation into light-sensitive photoreceptor phenotypes. (Invest Ophthalmol Vis Sci. 2010; 51: 1181-1189) DOI: 10.1167/iovs.09-4385
U2 - 10.1167/iovs.09-4385
DO - 10.1167/iovs.09-4385
M3 - Article
SN - 1552-5783
VL - 51
SP - 1181
EP - 1189
JO - Investigative Ophthalmology & Visual Science
JF - Investigative Ophthalmology & Visual Science
IS - 2
ER -