Abstract
Purpose :
Mutations in the CRB1 gene are amongst the leading causes for Retinitis Pigmentosa, however the exact function and localisation of the CRB1 protein in human retina is still not clarified. In our endeavour to elucidate CRB1 biology and the impact of CRB1 mutations, we use and study human induced pluripotent stem cell derived retinal organoids generated from control persons and affected patients.
Methods :
Human iPSC cells were generated from control persons and a pair of siblings with the CRB1 mutation C948Y. Subsequently, retinal organoids were differentiated according to an adapted protocol based on Zhong et al 2014. Organoids were either fixed or analysed at different time points (d40- d190). For immunofluorescence, samples were cryo-sected and stained with respective antibodies. For electron microscopy, cells were fixed at d190, embedded in araldite and later observed under an electron microscope. Ca2+ Imaging was performed at d200 after preincubation with Fura-2 calcium dye.
Results :
Presence of all human retinal cell as well as the inner and outer limiting membranes could be verified by immunofluorescence staining. Further, we could demonstrate the existence of an outer plexiform synapse layer with photoreceptor- bipolar cell synapses. Starting from day 40 of differentiation CRB1 could be found apical from the ZO-1 positive- outer limiting membrane in close apposition to the developing photoreceptor inner and outer segments. Furthermore, we could demonstrate the presence of connecting cilia as well as starting outer segment formation via electron microscope. Light sensitivity of organoid photoreceptors was shown via calcium imaging.
Conclusions :
All together, we could state the morphological hallmarks and verify the functionality of the retinal organoids derived from human iPSC. We could describe CRB1 protein expression during the retinal organoid development and set the groundwork for a thorough comparison of unaffected controls with retinal organoids obtained from patients with Retinitis Pigmentosa caused by CRB1 mutations.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.