Abstract
Purpose :
Axonal injury of retinal ganglion cells (RGCs) in the optic nerve head is an important early event in glaucomatous neurodegeneration. The molecular events leading to RGC axon degeneration, however, remain largely unclear due to the lack of human models to monitor this dynamic process. Human pluripotent stem cell-derived RGCs are important in vitro tool to study axonal degeneration and therapeutic drug development, but these applications require accurate and robust differentiation paradigms. We showed cell-based in vitro assays to study pathological features of glaucoma using human embryonic stem cell (hESC)-derived RGCs generated at high efficiency and fidelity.
Methods :
We generated RGCs from Brn3b-mCherry-H1 hESC line via two distinct directed differentiation methods and assessed differentiation efficiency and fidelity by comparing their scRNA-seq transcriptomic profiles to human fetal and adult RGCs. RGCs were then plated onto a microfluidic device to assess axonal degeneration using mechanical and chemical injuries. Following treatment, axon morphology was quantified, and changes in axonal transport were assessed by axonal trafficking of mitochondria through time-lapse imaging.
Results :
scRNA-seq analysis revealed the existence of three major cell classes with distinct molecular signatures, including progenitor, transitioning, and post-mitotic neuronal cells. Each method exhibited unique expression profiles along the differentiation trajectory. The neuronal clusters mapped robustly to human fetal RGCs, indicating high fidelity and specificity of the protocol for selective differentiation towards RGC-specific lineage. RGCs grown in a microfluidic device respond to different stressors with axonal degeneration, ascertained by changes in the morphology and axonal transport compared to controls.
Conclusions :
In this study, we performed a comparative single cell transcriptomic analysis of hESC-derived RGCs and a functional in vitro model of stress induced RGC axon degeneration leading to morphological and functional deficits in axons, mimicking some aspects of the phenotypes observed during the progression of glaucomatous neurodegeneration. The study will enhance our understanding of stem cell-derived RGC development and differentiation and the microfluidics model can support drug screening to identify targets that rescue neurodegenerative phenotypes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.