Enhanced induction of RPE lineage markers in pluripotent neural stem cells engrafted into the adult rat subretinal space.
OBJECTIVE
To investigate the differentiation of rat neural stem cells (rNSCs) into cells of retinal pigment epithelial (RPE) lineage both in vitro and in vivo, after subretinal transplantation into normal rats and in a sodium iodate (NaIO(3)) model of RPE loss.


METHODS
rNSCs prepared from the cortex of embryonic day (E)14 Fisher F344 rats were cocultured with different concentrations of vasoactive intestinal peptide (VIP), adult rat RPE cells, or neurosensory retina (NSR) for 5 days. Cell morphology and expression of RPE-specific markers (cytokeratin, CD68, microphthalmia-inducing transcription factor [MITF]) were studied. Additional antibodies used to characterize the rNSCs were markers for stem cells (nestin), immature neurons (betaIII-tubulin), astrocytes (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (Rip). In in vivo studies, 10(6) green fluorescent protein [GFP]-labeled rNSCs were injected subretinally in either normal adult Lewis rats or NaIO(3)-treated rats (70 mg/mL NaIO(3) administered intravenously 7 days before transplantation).


RESULTS
In vitro VIP-treated rNSCs changed from round cells to glia-like cells with processes that stained for both GFAP and nestin. In addition, small clusters of flattened, polygonal cells with an epithelial-cell-like shape that stained for cytokeratin and CD68 were observed. Coculture of rNSCs with RPE cells, but not with NSR, also led to cells of this phenotype. After transplantation, nestin(+) and GFP(+) rNSCs were visible subretinally as a transplant. In addition, more than 50% of transplanted rNSCs were cytokeratin(+) and CD68(+).


CONCLUSIONS
Very few rNSCs differentiate in vitro into epithelial-like cells that express RPE-specific markers. In vivo, this differentiation is remarkably enhanced after subretinal engraftment. Thus, transplantation of NSCs into the subretinal space may be a therapy for retinal diseases involving an RPE abnormality.
Enzmann
Volker
V
Department of Ophthalmology and Visual Sciences, University of Louisville, 301 E. Muhammad Ali Boulevard, Louisville, KY 40292, USA. volker.enzmann@louisville.edu
Howard
Russell M
RM
Yamauchi
Yasuyuki
Y
Whittemore
Scott R
SR
Kaplan
Henry J
HJ
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis*; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D018920:Coculture Techniques; D004268:DNA-Binding Proteins / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007452:Iodates; D007633:Keratins / Q000378:metabolism; D051739:Microphthalmia-Associated Transcription Factor; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D011917:Rats, Inbred Lew; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D012160:Retina / Q000601:surgery; D033581:Stem Cell Transplantation; D014157:Transcription Factors / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D004268:DNA-Binding Proteins; D005904:Glial Fibrillary Acidic Protein; D007452:Iodates; D007633:Keratins; D051739:Microphthalmia-Associated Transcription Factor; D011506:Proteins; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D014157:Transcription Factors; D014404:Tubulin; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C497236:Mitf protein, rat; C506249:RIPK1 protein, human; C032285:sodium iodate