Molecular Characterization of Human Retinal Progenitor Cells
Scott G Schmitt 1,
Unber Aftab 1,
Caihui Jiang 1,
Stephen Redenti 1,
Henry Klassen 2,
Erik Miljan 3,
John Sinden 3,
and
Michael J. Young 4*
1 Ophthalmology, The Schepens Eye Research Institute, Boston, Massachusetts, United States
2 Ophthalmology, University of California, Irvine, Orange, California, United States
3 ReNeuron Ltd., Guildford, Surrey, United Kingdom
4 Ophthalmology, The Schepens Eye Research Institute, 20 Staniford Street, Boston, Massachusetts, 02114, United States
* To whom correspondence should be addressed. E-mail: mikey{at}vision.eri.harvard.edu.
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Abstract |
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Purpose: 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/gene expression and neurotransmitter-stimulated calcium dynamics.
Methods: hRPCS were isolated from human retina of 14-18 weeks gestational age (G.A.) and expanded in vitro. Immunoblot, microarray and immunocytochemistry (ICC) assays were performed on undifferentiated hRPCs and those co-cultured with mouse retinas for two weeks. Cell function was assessed using calcium imaging.
Results: Our results from ICC showed a gradual decrease in the percentages of KI67, SOX2, and vimentin positive cells from passage one (P1) to P6, while 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. Upon 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: We conclude that 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.
Key Words:
Ca2+ channels, retinal cell culture, microarray, co-culture, human retinal progenitor cells
