Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells

Abstract

ABSTRACT.Purpose: The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. Methods: The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. Results: The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE ⁄ CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing b-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Conclusion: Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Derivation of Human Differential Photoreceptor-like Cells from the Iris by Defined Combinations of CRX, RX and NEUROD

“…A portion of purely isolated IPE cells of rodents, especially nestin-positive IPE cells, differentiated into multiple neuronal cell types, pan-neural marker- expressing cell types and retina-specific cell types without genetic manipulation. On the other hand, it has been shown that retinal stem cells are not present in the human iris [17], [18]. The present study demonstrates that human iris cells expressed stem cell markers such as nestin, N-cadherin, Sox2, Musashi-1 and Pax6.…”

A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain

“…The PR was carefully dissected from the corpus vitreum, to which it stayed attached after the CB was removed. A single-cell suspension was prepared by incubating the tissue in an enzyme solution, followed by careful trituration (73,74).…”

“…The primary cell suspensions from CB, iris, SVZ and vitreous samples were plated in a defined sphere-promoting medium, as we have previously described (73,74,76). Spheres were supplemented with growth factors twice a week, and passaged every two weeks.…”

Does the Adult Human Ciliary Body Epithelium Contain “True” Retinal Stem Cells?

“…Several studies have shown that CE spheres do express a range of immature neural and retinal markers [ 15 , 27 , 30 , 34 ]. Importantly, we found that the spheres contain two distinct populations of cells: one Nestin + and one Claudin-1 + , while no double positive cells were detected ( Figure 1(b) ) [ 32 ]. This suggests that, in contrary to the conclusion drawn by Cicero et al, CE-derived spheres consist of a homogenous population of ciliary epithelial cells, they contain both epithelial cells and cells with a more neural progenitor-like phenotype.…”