Generation of light-sensitive photoreceptor phenotypes by genetic modification of human adult ocular stem cells with Crx.
OBJECTIVE
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.
Jomary
Catherine
C
Clinical Neurosciences Division, University of Southampton, South Laboratory and Pathology Block, Southampton General Hospital, Southampton, United Kingdom. c.jomary@soton.ac.uk
Jones
Stephen E
SE
Lotery
Andrew J
AJ
D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D006152:Cyclic GMP / Q000378:metabolism; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology*; D018014:Gene Transfer Techniques; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008027:Light; D055537:Light Signal Transduction / Q000502:physiology*; D010641:Phenotype; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology* / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D015534:Trans-Activators / Q000235:genetics*
D015415:Biomarkers; D018398:Homeodomain Proteins; D015534:Trans-Activators; C109288:cone rod homeobox protein; D006152:Cyclic GMP