Prospective Identification and ex vivo Expansion of Retinal Progenitor Cells (RPCs) by Aldehyde Dehydrogenase (ALDH) Activity and in Co–Culture With Brain–Derived Endothelial Cells (BDECs)

Abstract

Abstract: : Purpose: The utility of retinal progenitor cell transplantation is hampered by the difficulty in obtaining therapeutically–sufficient numbers of these cells. Here, we explored novel methods to 1) identify populations of RPCs based on their ALDH activity and 2) expand these populations in transwell co–culture with BDECs. Methods: Tissue was dissected from the ciliary marginal zone and ciliary epithelium of neonatal rat and mouse retinas, dissociated with trypsin (0.25%) and maintained in culture medium supplemented with FGF2. After 7 days, many cells (putative RPCs) formed neurosphere colonies. ALDH staining: Cells from dissociated neurospheres were stained with BODIPY aminoacetaldehyde (Aldefluor), a fluorescent substrate for ALDH. Cells were sorted on a fluorescence activated cell sorter (FACS) based on intensity of fluorescence and side scatter (SSC). Sorted cells were reseeded onto 12–well plates at a density of 5x10³ cells/well to assess their differential capacities for expansion. After 3 wk colonies were visually counted. BDEC co–culture: BDECs were isolated from neonatal rat and mouse brain vessels and maintained in complete endothelial cell medium. For transwell studies, cells were reseeded onto 6–well plates and grown to 60% confluence. RPC transwells were inserted and maintained in medium containing BDECs+FGF, BDECs alone or FGF alone for 4 days. Cells then were dissociated and reseeded in RPC medium with FGF before visually counting. Results: FACS sorting revealed distinct cell populations. A large number of cells were brightly–positive for ALDH (ALDH^br), and these were further sorted into two distinct groups based on SSC (SSC^hi and SSC^lo). After reseeding, ALDH^brSSC^hi cells formed larger and more–rapidly growing colonies (23+12 vs. 1+1.73 colonies/10x field). In transwell cultures, cells proliferated preferentially in medium with BDECs+FGF, forming larger neurospheres than with FGF or BDECs alone. Two days after reseedeing on 12–well plates, there were 16+7 colonies/10x field with BDECs+FGF, 10+7 with BDECs alone, and 7+3 with FGF alone. Conclusions: As with other progenitor cell populations, RPCs have high ALDH activity. This can be useful for prospectively identifying RPCs. Furthermore, the number of RPCs can be preferentially expanded when exposed to BDECs. These strategies can be useful in obtaining adequate numbers of RPCs for transplantation.

Keywords: retina • vascular cells • flow cytometry