Molecular characterization of human retinal progenitor cells.
OBJECTIVE
To examine the molecular profile of fetal human retinal progenitor cells (hRPCs) expanded in vitro and those grown in a co-culture system with mouse retina through the analysis of protein and gene expression and neurotransmitter-stimulated calcium dynamics.


METHODS
hRPCS were isolated from human retina of 14 to 18 weeks gestational age (GA) and expanded in vitro. Immunoblot, microarray, and immunocytochemistry (ICC) assays were performed on undifferentiated hRPCs and those co-cultured with mouse retinas for 2 weeks. Cell function was assessed by using calcium imaging.


RESULTS
The ICC results showed a gradual decrease in the percentages of KI67-, SOX2-, and vimentin-positive cells from passages (P) 1 to P6, whereas a sustained expression of nestin and PAX6 was observed through P6. Microarray analysis of P1 hRPCs showed the expression of early retinal developmental genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2, HES5, GNL3, OTX2, DACH1, SIX6, and CHX10 (VSX2). At P6, hRPCs continued to express VIM, KI67, NES, PAX6, SOX2, GNL3, and SIX6. On co-culture, there was a significant increase in the expression of MKI67, PAX6, SOX2, GNL3, SIX3, and RHO (rhodopsin). Calcium imaging showed a functional response to excitatory neurotransmitters.


CONCLUSIONS
Fetal-derived hRPCs show molecular characteristics indicative of a retinal progenitor state up to P6 (latest passage studied). They show a progressive decrease in the expression of immature markers as they reach P6. These cells are functional, respond to excitatory neurotransmitters, and exhibit changes in expression patterns in response to co-culture with mouse retina.
Schmitt
Scott
S
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Aftab
Unber
U
Jiang
Caihui
C
Redenti
Stephen
S
Klassen
Henry
H
Miljan
Erik
E
Sinden
John
J
Young
Michael
M
D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003598:Cytoskeletal Proteins / Q000378:metabolism; D053686:Fetal Stem Cells / Q000166:cytology* / Q000502:physiology; D019084:Fluorescent Antibody Technique, Indirect; D005865:Gestational Age; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012160:Retina / Q000166:cytology* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017299:Rod Opsins / Q000378:metabolism
D000943:Antigens, Differentiation; D015415:Biomarkers; D003598:Cytoskeletal Proteins; D019394:Ki-67 Antigen; D009419:Nerve Tissue Proteins; D017299:Rod Opsins; C095853:short-wavelength opsin; D002118:Calcium