Abstract
Purpose: :
Photoreceptor degeneration is a leading cause of blindness in the western world. The discovery of iPS cells and their potential use for disease modelling means developmentally relevant methods for the differentiation of mature photoreceptors are required. We sought to investigate photoreceptor differentiation in ES cell-derived 3D retinal cultures to determine if this method models in vivo photoreceptor development and ultimately produces structurally mature photoreceptors.
Methods: :
Mouse ES cell lines (CCE and 129) were differentiated using a 3D culture system similar to that described by Eiraku et al. 2011. The main difference was that embryoid bodies were retained and the retinal neuroepithelia were not cultured separately. This enabled photoreceptors to develop at the apical edge of the neural epithelia and project into the luminal cavity of the embryoid body. Photoreceptor differentiation was examined by RT-PCR and immunocytochemistry over the 35 day culture period and the structural development of photoreceptors was assessed by electron microscopy.
Results: :
Neural epithelia were present from day 9 in culture and displayed polarised cell proliferation, similar to the retinal neuroblastic layer, at day 18. Following 20 days of culture, Crx+ photoreceptor precursors were present and by day 26 rhodopsin+ and recoverin+ cells maintained an ‘ONL-like’ layer at the apical edge of the neural epithelia. Cralbp+ müller glial processes spanned this layer and maintained ZO-1+ adherens junctions at the outer edge, reminiscent of the OLM. Later stage photoreceptor markers, such as rod transducin, were also present from day 28 of culture. Ultrastructural analysis demonstrated the presence of ‘inner-like’ segments from day 28 onwards, similar to the postnatal retina. However, ‘outer-like’ segments were not observed.
Conclusions: :
Photoreceptor development in 3D ES cell-derived cultures paralleled in vivo development and ES cell-derived photoreceptors were ultrastructurally comparable to postnatal photoreceptors. However, the organisation of these cells was compromised following 35 days of culture; consequently it was unclear whether these cells could progress to form ‘outer-like’ segments. Therefore further modifications to increase the survival of these cells may be required to create an in vitro model for photoreceptor degeneration.
Keywords: photoreceptors • differentiation • retinal culture