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Human iPS-derived Retinal Pigment Epithelium (RPE) Cells Exhibit Transport, Membrane Potential, Polarized VEGF Secretion and Gene Expression Pattern Similar to Native RPE

From the May 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Complimentary to another article involving the retina, also featured in the May edition of Stem Cells1, the potential for induced pluripotent stem cells (iPSC) to generate replacement retinal components is further demonstrated in an article by Kokkinaki et al.2 from Georgetown University in Washington DC, which reports that iPSCs can be differentiated into retinal pigmented epithelium (RPE) that can perform many of the normal functions of native RPE. The health of the light sensitive photoreceptors that reside at the back of the retina are dependent on a functional RPE, in fact the two cell types are interdependent. Within the eye, the apical membrane of the RPE cells face the outer segments of the photoreceptors and not only phagocytose shed photoreceptor outer segments but perform a number of other functions including the release of growth factors and isomerisation of retinal from the phototransduction cycle. Various forms of retinal disease require the replacement of dysfunctional RPE with newfunctional RPE, such as age-related macular degeneration (AMD), where impaired RPE function in turn causes the death of macular photoreceptors. Many reports have shown the facile generation of RPE from both human embryonic stem cells (hESC) and induced pluripotent stem cells (RPE), making these cell types an attractive source of replacement RPE. 3,4

The generation of RPE from pluripotent cell types is not new – many reports have now shown that RPE can be derived quite easily from hESCs and human iPSCs (hiPSC). In fact, the similarity of hESC-derived RPE to native RPE has been demonstrated by various groups. hESC-derived RPE over time can acquire the characteristic gene expression profile, phagocytose, polarise and secrete vascular endothelial growth factor (VEGF), just like normal RPE. These results have led to the approval of clinical trials in America by the Food and Drug Administration (FDA) and forthcoming preclinical trials in the UK by various regulatory bodies to test the potential of hESC-derived RPE for the restoration of this cell type in humans. The use of hiPSC to this end would be even more desirable, providing the opportunity for patient-specific cell replacement and bypassing the obvious immunological and ethical issues associated with the use of hESC. Kokkinaki et al.2 expand on what is already known by assessing the ion channel transport activity, membrane potential and long term integrity of hiPSC-derived RPE.

Using the IMR90-4 WiCell hiPSC line, the authors generated RPE by differentiating hiPSCs as aggregates in suspension in the presence of nicotinamide and Activin-A for 4 weeks and then transferring the cells into adherent culture conditions for a further few weeks. Analysis of gene expression by QPCR and various RPE markers following immunostaining of differentiating cells confirmed the emergence of RPE-specific antigens and genes (MITF, OTX2, RPE65) within cultures, with approximately 90% of cells becoming pigmented by the 8th week of differentiation and which displayed typical hexagonal RPE morphology. The authors demonstrate that 56 of 89 genes recently validated to be part of the RPE molecular signature5 show similar expression in hiPSC-generated RPE compared with native RPE. In hiPSC-derived RPE cultures grown on transwell inserts the authors investigated cellular polarity by assessing the localisation of Na+/K+ ATPase on the apical surface and by measuring the level of VEGF secretion by performing VEGF ELISA assays on media collected from the upper and lower surface of the cell monolayer. These experiments confirmed the localisation of Na+/K+ ATPase to the apical surface and VEGF secretion from the basal surface, akin to native RPE. The ability of up to 90% of hiPSC-RPE cells to internalise fluorescent latex beads also demonstrated their phagocytic capacity. Kokkinaki et al.2 then performed whole cell patch clamp electrophysiology on hiPSC-RPE cells which confirmed active voltage gated Na+ and K+ ion channels.

Importantly, the authors elaborate upon some of the shortcomings of hiPSC-derived RPE cells. Kokkinaki et al.2 observed a significant decrease in cell growth over passages 5 to 7 so went on to determine whether there were any changes in telomere length with increasing passage number by detecting the relative telomere repeat copy number by QPCR. The results of this work revealed that rapid telomere shortening occurs in hiPSC-RPE populations from passage 5 onwards, alongside increased expression of the cell cycle inhibitor p21 and cell growth arrest. Indicators of high DNA chromosomal damage were also detected. These results all indicate accelerated senescence in hiPSC-derived RPE cells, which corroborates with findings in other hiPSC derivatives.6

This is particularly important result for the long term functionality of transplanted tissue. Although a number of studies now demonstrate that RPE derived from pluripotent cell types are capable of acting like native RPE in various ways - representing great achievement for the development of cell-based restorative therapy for the treatment of blindness, one cannot ignore the results presented herein which indicate that, at least in this study, hiPSC-derived RPE after multiple passage exhibit rapid telomere shortening and DNA damage. This may indeed be the underlying reason for the rapid loss of grafted RPE reported in some studies, observed as few as two weeks following transplantation. The underlying causal factors remain unclear, however the authors implicate that this may arise due to the reprogramming process itself. It is also unknown whether utilising earlier passage cells, for example at passage three prior to significant telomere shortening, will achieve better results for long term functionality, but will the tumorigenic potential of such cells then be under question? Advanced Cell technology are currently recruiting patients with advanced dry AMD and Stargardt's Macular Dystrophy to commence clinical trials using hESC-derived RPE and Pete Coffey at the London project to cure blindness in collaboration with Pfizer Regenerative Medicine and UCL is leading the way towards similar trials in patients in the UK. These tightly regulated preclinical studies alongside those currently recruiting will no doubt shed light on the safety of this procedure for use in humans.



1 Zhou et al. Differentiation of Swine iPSC into Rod Photoreceptors and Their Integration into the Retina. Stem Cells, May 2011.

2 Kokkinaki et al. Human Induced Pluripotent Stem-Derived Retinal Pigment Epithelium (RPE) Cells Exhibit Ion Transport, Membrane Potential, Polarized Vascular Endothelial Growth Factor Secretion, and Gene Expression Pattern Similar to Native RPE. Stem Cells 2011, 29(5):825–835.

3 Rowland et al. Pluripotent human stem cells for the treatment of retinal disease. J Cell Physiol. 2011 [Epub ahead of print] doi: 10.1002/jcp.22814.

4 Vugler et al. Embryonic stem cells and retinal repair. Mech Dev. 2007, 124(11-12):807-29.

5 Strunnikova et al. Transcriptome analysis and molecular signature of human retinal pigment epithelium. Hum Mol Genet. 2010, 19(12):2468-86.

6 Feng et al. Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence. Stem Cells 2010, 28(4):704-12.


See a related original article from this month’s edition by Zhou et al. and this paper’s commentary on the Stem Cells Portal.