Abstract
Purpose:
In light of recent advances in stem cell biology and prospects of cell transplantation in the field of ophthalmology, it is increasingly important to characterize retinal cells, especially in the early stages of eye development. Our primary objective is to define surface markers to enable isolation of pure cell populations in the human fetal retina and human embryonic stem cell (ES)-derived retinal tissue for further study.
Methods:
To demarcate unique cell types in the developing human retina, we screened a fetal retina at 20 weeks gestation, with a commercially available cell surface marker antibody panel. Dissociated retinal cells were co-labeled with CD133 and each of 251 monoclonal antibodies, and analyzed by flow cytometry. CD133, a marker of progenitors and photoreceptors, was employed to aid in defining populations marked by panel antibodies. Dissociated ES-derived retinal cells were analyzed for expression of a subset of markers present in fetal retina. In addition, immunofluorescence microscopy was used to examine the pattern of expression of many of the antibodies in fetal retina 15 to 21 fetal weeks in age.
Results:
Of the surface markers analyzed by flow cytometry, 112 were expressed in some cells in the human fetal retina. The 55 markers most frequently expressed on retinal cells exhibited one of eight different patterns of expression when analyzed along with cell size and CD133 expression. Patterns detected by antibodies to CD73, CD15, and CD44 are consistent with patterns previously described in the murine retina, a photoreceptor precursor marker, and early and late progenitor markers, respectively. Furthermore, 49-day-old ES-derived retina exhibited expression of 45 of 50 antigens expressed in fetal retina, suggesting that those not expressed are displayed on more mature cell types. Immunofluorescence analysis demonstrated that some surface markers differed in their expression levels from central to peripheral retina indicative of cells of differing maturational states.
Conclusions:
Commonly available cell surface antigens can be used to separate disassociated single cell suspensions of retinal cells into distinct subpopulations. These markers may enable purification of retinal cell types from heterogeneous populations, including photoreceptor precursors, ganglion cells, and interneurons from both human fetal retina and ES-derived retina.
Keywords: 688 retina •
698 retinal development •
529 flow cytometry