The Anatomy of RPE Production From Human Embryonic Stem Cells

Abstract

Purpose: : To use an anatomical approach in order to gain insights into the genesis and expansion of retinal pigment epithelium (RPE) cells derived from human embryonic stem cell (HESC) cultures.

Methods: : Following super-confluent growth of HESC on feeders, pigmented foci were dissected free and expanded as monolayers on matrigel. Specimens were either processed for electron microscopy, immunocytochemistry, western blot or RTPCR, with the ARPE19 cell line used for comparison. The influence of dissociation and extracellular matrix on HESC-derived RPE cells was also examined by generating single cell suspensions from monolayers.

Results: : In HESC cultures, pigmentation was preceded by the expression of a pre-melanosomal marker, with the emergence of melanosomes in individual cells corresponding to elevated OTX2. Pigmented cells exited cell cycle and were surrounded by non-pigmented cells positive for both OTX2 and the RPE-associated keratin 8, or βIII tubulin. The presence of RPE65 in these preparations was confirmed at both mRNA and protein level. Following placement of pigmented foci onto matrigel, RPE cells de-pigment, re-enter the cell cycle and express keratin 8 at the advancing edge of monolayers. Keratin 8 expression is subsequently lost as the cells mature, re-pigment and exit the cell cycle (determined by Ki67 and BrdU labelling). The RPE cells maintained expression of OTX2 throughout and became polarised, with apical Na⁺/K⁺ ATPase, microvilli, Cilia (typically 1-2 per cell), and connexon channels. Upon removal from the monolayer environment, isolated HESC-derived RPE displayed a more neural phenotype, characterised by the extension of processes and the upregulation of βIII tubulin, a phenomenon that was potentiated by laminin.

Conclusions: : RPE cells arise from their progenitors in cultures of HESC and can rapidly exit the cell cycle. The RPE can then act as their own precursors, expanding and differentiating as a coherent monolayer. This structure develops in the absence of retinal cues and displays features consistent with foetal RPE. Importantly, dissociation of these cells into a cell suspension can lead to phenotypic alterations characteristic of the trans-differentiation seen in RPE from lower vertebrates. Although this phenomenon may be utilised to generate other clinically relevant retinal cell types from HESC-derived RPE, the use of cell suspension-derived cell lines for transplantation therapy may prove problematic.

Keywords: retinal pigment epithelium • transcription factors • NaK ATPase