PHOTORECEPTOR

New in vivo experimental data support the idea that replacement of lost photoreceptors by cell transplantation should in time represent a possible treatment option for affected patients, Dr Marius Ader, a principal investigator at the Centre for Regenerative Therapies, University of Technology, Dresden (CRTD), told delegates at the EURETINA Winter Meeting. Dr Ader reported on experiments at his institute and explained that while photoreceptor transplantation has been on-going for 20 years, technical advances had moved the field forward considerably over the recent five years. His team routinely isolate primary photoreceptor cells from the peak of rod generation in donor animals and, when GFP ('green fluorescent protein')- labelled, transplant the cells sub-retinally into recipient models. Results presented at the meeting showed green donor cells integrated in the outer nuclear layer with a morphology resembling mature photoreceptors.

Transplanted cells express photoreceptor specific markers, including recoverin. However, a key interest is to determine if transplanted cells can form outer segments. Dr Ader used an engineered reporter mouse in which rhodopsin is fused to the GFP label so that the signal represents the actual location of rhodopsin in the retina. Further modifications led to the development of a double reporter mouse which crossed a rhodopsin GFP mouse with an actin DsRed mouse, which, according to Dr Ader, allowed the researchers to see 'the red fluorescence in the cell body and GFP in the outer segment of photoreceptors'. The research group found that the double reporter allowed one 'to follow both the integration of photoreceptors and outer segment formation following transplantation using fluorescence microscopy without further staining procedures'.

[caption id='attachment_2262' align='alignright' width='225' caption='Integrated photoreceptors following transplantation into an adult mouse retina. While the cell body of the donor cells are labelled by DsRed (magenta) the outer segments are labelled by GFP (green) at the tip of the photoreceptor. INL: inner nuclear layer; IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; OS: outer segments'][/caption]

'You can then go even further and use antibodies against GFP and label them with gold particles and look at the ultra-structure of the outer segments to examine the discfilled outer segments,' he explained. Using a correlative light and electron microscopy technique, Dr Ader showed the meeting that the transplanted cells' outer segments are absolutely comparable with the wild-type endogenous outer segments, providing convincing evidence that transplanted cells integrated into the host retina and formed an outer segment. While such results were encouraging, Dr Ader knew that if the technology were to be of real clinical value then it would need to be demonstrated in a disease model. His team used the available RP p347s disease model which had an almost non-existent ONL at the time of transplantation. However, following treatment, clear healthy cells were detectable in the recipient disease model including ultra-structure imagery showing proper outer segments with connecting cilia. The transplanted precursor cells were able to mature and form disk filled outer segments.

The team pushed further to increase the rate and proficiency of the transplantation as they wished to focus on improving the low integration rate (Dr Ader showed the conference convincing data following transplantation of the CD73+ sorted cells demonstrating increased integration of photoreceptors. The team have additionally developed an improved protocol where around 10,000 such cells have been integrated in the retinas. He concluded his presentation by summarising the achievements of transplanting immature photoreceptors to allow for correct integration into the adult mammalian retina followed by the use of MACS enrichment by cell surface markers leading to increased integration. 'Such purification procedures may prove extremely important for the selection of in vitro generated stem-cell derived photoreceptors for transplantation studies and represent an indispensable prerequisite for possible future clinical applications'.