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Differentiation of Swine iPSC into Rod Photoreceptors and Their Integration into the Retina

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From the May 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Stem cell therapy remains one of the most promising options for restoration of the degenerative retina. Featured in the May edition of Stem Cells are two articles (Zhou et al.1 and Kokkinaki et al.2) which demonstrate that induced pluripotent stem cells (iPSC) can be differentiated into various components of the mature retina. These articles individually address two important considerations; the first, discussed herein, shows that iPSC-derived photoreceptors can integrate and start to develop features typical of morphological maturation following transplantation into the degenerative retina, and the other that retinal pigmented epithelium (RPE) generated from iPSC is capable of acting in a functional manner (see the link to this paper commentary on the Stem Cell Portal homepage). The light-sensitive photoreceptors reside in the outer nuclear layer (ONL) of the retina and are supported by the underlying RPE. The RPE transfers oxygen and nutrients to the photoreceptors from the choroidal blood supply and performs many functions essential for the health of the photoreceptors, including phagocytosis of shed photoreceptor outer segments and the removal of the waste products of the visual cycle. The article by Zhou et al.1 from the Department of Ophthalmology at Central South University in China and multiple collaborative centres at the University of Louisville, describes the differentiation of cells from a swine iPSC line into rod photoreceptors and their integration within a swine model of retinal degeneration.

Whilst the differentiation of pluripotent cell types towards a photoreceptor fate has already been demonstrated by multiple groups, the final step in the morphological maturation of these cells by formation of the outer segment has proven to be the greatest challenge. The membranous disks of the outer segment contain visual pigments necessary for phototransduction and are therefore an indicator of functional maturation. Following transplantation of embryonic stem cell (ESC) or iPSC-derived photoreceptor precursors, the vast majority of studies report the failure of grafted cells to develop an outer segment. Zhou et al.1 however, managed to achieve this final step. Photoreceptor cells were derived by differentiating iPSCs using a similar protocol to what has now been described by multiple groups to yield photoreceptor-like cells from pluripotent populations, with some modification. The authors antagonised bone morphogenetic pathway (BMP) and Wnt/β-catenin pathways and supplemented the cultures with insulin-like growth factor 1 (IGF-1) and neural supplements B27 and N2. Cells were differentiated first in suspension culture as embryoid bodies (EBs) and then under adherent conditions. The authors tested various substrates; adherent cultures were grown on poly-D-lysine coated plates with different concentrations of Matrigel solution or with laminin and fibronectin. After 21 days they found that 6% of the cells expressed rhodopsin, a terminal marker of photoreceptor differentiation, and a number of photoreceptor-associated mRNAs were upregulated (RHO, ARR3, RBP3, RECOVERIN). Importantly, a proportion of rhodopsin+ cells with a rounded cell body and which had formed rhodopsin-concentrated projections, reminiscent of the outer segment, were observed. Culture of cells on Matrigel was found to yield the greatest number of cells with a rounded soma and RHO and ROM1-rich outer segment-like extensions. This is an important result given the recent Nature paper by Eiraku et al. which demonstrates that mouse ESCs cultured in a three-dimensional (3D) Matrigel can differentiate towards a retinal lineage and self-organise into optic cup structures.3 This highlights to importance of the extracellular matrix and the way in which the cells are grown during differentiation in allowing them to reach their full phenotypic potential.

Iodoacetic acid treatment leads to selective loss of rod photoreceptors in the swine retina, leaving cone photoreceptors intact. Zhou et al.1 used this method to ablate the rod population in 6 week old pigs. They transfected differentiated iPSC with lentivirus coding for the RBP3 promoter (a photoreceptor differentiation marker which maintains its expression in mature photoreceptors) driving green fluorescent protein (GFP, achieving 44% transfection efficiency) and then assessed the integration of differentiated iPSC transplanted subretinally into rod-deficient eyes. After three weeks they detected RHO/GFP+ cells across the host retina with the majority found within the ONL, some of which had developed outer segment-like projections. Although the authors make assumptions about the widespread integration of grafted cells within retinae and go on to generate their calculations of cellular integration from this, they nonetheless estimate a 1% integration of grafted cells which is comparable to other work of this nature. However donor cell integration did not lead to any improvement in dark-adapted ERG.

The observation that swine iPSC-derived photoreceptor cells can form an outer segment, both in vitro and following transplantation into the rod deficient swine eye in vivo is novel – this achievement has not been reported following transplants of hESC-derived cells into various mouse models of retinal degeneration. Yet the development of an outer segment in integrated cells within the ONL did not lead to any improvement in host visual function which, ironically, has been reported following integration of donor cells in the ONL in mouse models of retinal degeneration - in the absence of any detectable donor outer segment formation. The only study which has demonstrated the integration and full morphological maturation and functionality of grafted cells (with outer segment formation) came from a report by MacLaren et al.4 in which grafted mouse Nrl-GFP positive postmitotic photoreceptor precursor cells subretinally into the mouse eye. It is interesting to note that the best results have been achieved following allogeneic transplants, when the donor species is matched to the host. One has to wonder – might this be the reason why many studies performing xenotransplantation of human cells into small animal models have been unable to achieve this final step? Maybe these results should prompt us to revisit the relevance of the animal models we are currently utilising.

The ability to generate functional replacement components for the degenerative retina relies heavily upon initial results that are generated following experiments which use small animal models of disease. There is certainly a degree of homology between rodents and humans, yet the organisms as a whole are very different. Can we ignore this fact? And might this then be the limiting factor in revealing the true potential of stem cell therapy for the retinal disease; the ability to test the replacement potential of donor tissues in more comparable host species? Certainly, proof-of-principle experiments to assess the tumorigenic potential and long term survival of grafted cells have their place in small animal models, but the use of more relevant models in the development of stem cell therapy for future translation to the clinic is likely to be of great informative value. Not only is the porcine retina anatomically much more similar to the human retina, but it contains a central streak that is rich in cone photoreceptors, reminiscent of the human macula, the region of the retina that is essential for fine visual acuity and the perception of colour. Swine models of photoreceptor degeneration, such as the rhodopsin transgenic pig model of human Retinitis Pigmentosa, already offer alternatives to more commonly used small animal models. Rodent models are certainly not ideal for assessing the efficacy of treatments that are designed to help patients with macular dystrophy, having a predominantly rod-enriched retina and lacking a cone-rich macular-like region. Would we therefore see different results after transplanting human cells into the swine eye? In addition, in vitro protocols used for photoreceptor generation cannot be ignored. In one study, swine neonatal retina was transplanted into the pig eye and although retinal grafts survived well, late stage photoreceptor markers were not observed.5 It is clear that not only is the animal model an indicator of the experimental outcome, but this work also highlights the importance of the ontogenetic stage of grafted cells, as has been previously demonstrated in the mouse.4

 

References

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 Eiraku et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 2011, 472:51–56.

4 Maclaren et al. Retinal repair by transplantation of photoreceptor precursors. Nature 2006, 444:203-207.

5 Ghosh et al. Transplantation of full-thickness retina in the rhodopsin transgenic pig. Retina. 2004, 24(1):98-109.

 

Also see the related original article from this month’s edition by Kokkinaki et al. and the paper commentary on the Stem Cells Portal.