Mapping function of whole-mount retinal organoids with LSFM

Session

Poster Session

Authors

Marina Cunquero (2), Helena Isla-Magrané (3), Gustavo Castro-Olvera (2), Jordi Soriano (1, 4), Maria Marsal (2), Nicolás Mateos (2), Maddalen Zufiaurre (3), Josep García-Arumí (3), Anna Duarri (3), Pablo Loza-Alvarez (2)

Affiliations

1. Departament de Física de la Matèria Condensada, Universitat de Barcelona
2. ICFO-Institut de Ciències Fotòniques
3. Ophthalmology Research Group, Vall d'Hebron Institut de Recerca (VHIR)
4. Universitat de Barcelona Institute of Complex Systems (UBICS)

Keywords

Retinitis Pigmentosa, Retinal Organoids, Light-sheet Fluorescence Microscopy, Calcium Dynamics, Optical Clearing

Abstract text

Cell therapy stands as a promising treatment avenue for retinal degenerative diseases, such as Retinitis Pigmentosa (RP), an age-related macular degeneration (AMD) disease. The transplantation of new generated photoreceptors imposes a challenge in acquiring mature, high-quality cells that seamlessly integrate and function within the diseased retinal environment. Retinal Organoids (RO) have been proven to recapitulate the structure and physiology of the tissue. They have a huge potential for disease modelling and personalized medicine, and can also be used as a source of precursor photoreceptors for cell therapy. In the pursuit of designing effective therapeutics and to advance our comprehension of RO maturation, here we aim at characterizing the structure and function of retinal neurons from RO derived from human induced pluripotent stem cells (ihPSC). This characterization is crucial for pinpointing the optimal timing for harvesting photoreceptors, a pivotal step in the subsequent implantation process. RO are complex 3D structures, containing highly dense cell populations, and thus, they are opaque making characterization difficult. Moreover, they are highly variable imposing the challenge that a full picture of the entire specimen is needed. That is why this study aims at characterizing whole-mount RO in a 3D and dynamic context. To do so, we tracked the calcium dynamics in 3D with light-sheet fluorescence microscopy (LSFM). Then, we used optical clearing methods in combination with cell-type specific immunohistochemistry assays for identifying the distinct cell types conforming to the retinal layers.

For our study, we used RO derived from healthy and RP donors. Viral vectors encoding for GCaMP6s were employed to visualize changes in calcium influx. We tracked calcium activity in 3D with a custom-made inverted Selective Plane Illumination Microscope (iSPIM). This optical configuration allowed fast tracking of dynamics (16 Z-planes at 5 Hz) occurring on a volumetric scale (800 x 800 x 0.1 mm) with minor photo-toxic effects. Quantitative analysis of the functional communities of neurons based on transfer entropy statistics are currently being performed. This will provide the pattern and frequency of calcium waves in the neuronal network and that ultimately will serve to map function in 3D. After the in vitro measurements, we imaged the structure of mature ROs in a whole-mount configuration by using an optimized optical clearing method based on FluoClear BABB, combined with enhanced antibody permeabilization. This allowed to identify the morphology and population of the three neuron-path that provide the direct route for visual information transmission: cone and rod photoreceptors, bipolar and ganglion cells. 

Our LSFM system demonstrates applicability in studying rapid events within millimetre-range samples with cellular resolution. In the future, the methodology presented in this study has the potential to provide insights into the temporal, spatial, and functional organization of retinal cells within the organoid.