Characterization of dorsal root ganglia sensory neurons from human pluripotent stem cells and their application for developing therapies to treat Friedreich Ataxia

Abstract

Sensory neurons of the dorsal root ganglia (DRG) are the primary responders to stimuli inducing feelings of touch, pain, temperature, vibration, pressure and muscle tension. They consist of multiple subpopulations based on their morphology, molecular and functional properties. Our understanding of DRG sensory neurons has been predominantly driven by rodent studies and using transformed cell lines, whereas less is known about human sensory DRG neurons simply because of limited availability of human tissue. Although these previous studies have been fundamental for our understanding of the sensory system, it is imperative to profile human DRG subpopulations as it is becoming evident that human sensory neurons do not share identical molecular and functional properties found in other species. Furthermore, there is a wide range of diseases and disorders that directly/indirectly cause sensory neuronal degeneration or dysfunctionality, such as Friedreich Ataxia (FRDA) disease. FRDA is an autosomal recessive disease characterized by degeneration of DRG proprioceptive sensory neurons, which is due to a low level of the mitochondrial protein Frataxin (FXN). Having an in vitro source of human DRG sensory neurons, such as from human pluripotent stem cells (hPSC), is paramount for studying their development, unique neuronal properties and for accelerating regenerative therapies to treat sensory neuropathies, including FRDA. To this end, in this thesis, we generated DRG sensory neurons both from hESC and induced pluripotent stem cells (iPSC) from FRDA patients, characterizing DRG sensory neurons subtypes. Our results show that our protocol gives rise to heterogenous subpopulations of sensory neurons, that include nociceptors, mechanoreceptors and proprioceptors, as indicated by immunostainings and Q-PCR analyses. We also observed that the generation of the subtypes is a dynamic process, with expression of different markers arising at different time points in cultures during differentiation. In summary, these data provide an in vitro platform to discover the unique gene expression profile of sensory neuronal cell types that may exist within the human DRG and offer also the possibility to use this platform to model sensory neurons-related diseases in vitro.

Another aim of this thesis was to interrogate the ability of hPSC-derived sensory neurons to survive, differentiate and integrate in vivo. Transplantations of hPSC-derived sensory neural progenitors, both from hPSCs and FRDA iPSCs, were performed in the DRG of adult rats and tissue analyses were performed at 2-8 weeks post-transplantation. Our results showed survival of transplanted donor cells, within and surrounding the DRG and, most importantly, donor cells expressed markers of DRG sensory neurons and glia, demonstrating their capacity to differentiate to sensory neuronal and glial lineages in vivo. These novel studies start to address the possibility of developing cell replacement therapies for treating neurodegeneration occurring within the peripheral sensory nervous system, particularly in FRDA.

Lastly, we explored the therapeutic application of nanoparticles to deliver Frataxin-expression plasmid into hPSC and FRDA iPSC-derived sensory neurons. We tested several nanoparticle types that varied in their composition properties, sizes and charge to identify the most effective nanoparticles that are capable of being taken up by sensory neurons and deliver plasmid. Our preliminary results were promising for showing potential therapeutic use of nanoparticles as delivery system.

In conclusion, our data provide new insights for using hPSC as a model to study human DRG development, phenotypic and functional properties of human sensory neuronal subtypes and for evaluating advanced alternative therapies to treat DRG sensory-related disorders, particularly FRDA.