Facile isolation and the characterization of human retinal stem cells

doi:10.1073/pnas.0401596101

Facile isolation and the characterization of human retinal stem cells

^*Departments of Medical Genetics and Microbiology, University of Toronto, Toronto, ON, Canada M5S 1A8; ^‡Unit of Oculogenetic, Hôpital Ophtalmique Jules Gonin, 1004 Lausanne, Switzerland; ^§Department of Zoology, Miami University, Oxford, OH 45056-1400; and ^¶Department of Molecular and Medical Genetics, University of Toronto, and Programs in Development and Genetics, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8

Edited by Thaddeus P. Dryja, Harvard Medical School, Boston, MA (received for review March 5, 2004)

Abstract

This study identifies and characterizes retinal stem cells (RSCs) in early postnatal to seventh-decade human eyes. Different subregions of human eyes were dissociated and cultured by using a clonal sphere-forming assay. The stem cells were derived only from the pars plicata and pars plana of the retinal ciliary margin, at a frequency of ≈1:500. To test for long-term self-renewal, both the sphere assay and monolayer passaging were used. By using the single sphere passaging assay, primary spheres were dissociated and replated, and individual spheres demonstrated 100% self-renewal, with single spheres giving rise to one or more new spheres in each subsequent passage. The clonal retinal spheres were plated under differentiation conditions to assay the differentiation potential of their progeny. The spheres were produced all of the different retinal cell types, demonstrating multipotentiality. Therefore, the human eye contains a small population of cells (≈10,000 cells per eye) that have retinal stem-cell characteristics (proliferation, self-renewal, and multipotentiality). To test the in vivo potential of the stem cells and their progeny, we transplanted dissociated human retinal sphere cells, containing both stem cells and progenitors, into the eyes of postnatal day 1 NOD/SCID mice and embryonic chick eyes. The progeny of the RSCs were able to survive, migrate, integrate, and differentiate into the neural retina, especially as photoreceptors. Their facile isolation, integration, and differentiation suggest that human RSCs eventually may be valuable in treating human retinal diseases.

Footnotes

↵ † B.L.K.C. and B.A. contributed equally to this work.
↵ ^∥To whom correspondence may be addressed at: Unit of Oculogenetic, Hôpital Ophtalmique Jules Gonin, 15, Av. de France, 1004 Lausanne, Switzerland. E-mail: yvan.arsenijevic{at}chuv.hospvd.ch. ^**To whom correspondence may be addressed at: Department of Medical Genetics and Microbiology, University of Toronto, 1 Kings College Circle, Toronto, Canada M5S 1A8. E-mail: derek.van.der.kooy{at}utoronto.ca.
Author contributions: Y.A. and D.v.d.K. designed research; B.L.K.C., B.A., T.I., K.D.R.-T., J.R.S., and Y.A. performed research; R.R.M. contributed new reagents/analytical tools; B.L.K.C., B.A., T.I., K.D.R.-T., J.R.S., R.R.M., and Y.A. analyzed data; and B.L.K.C. and D.v.d.K. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: RSC, retinal stem cell; NSC, neural stem cell; RPE, retinal pigmented epithelium; EGFP, enhanced GFP; EGF, epidermal growth factor; F+H, fibroblast growth factor 2 plus heparin; E+F+H, EGF plus fibroblast growth factor plus heparin; MAP2, microtubule-associated protein 2.