Frataxin gene editing rescues Friedreich’s ataxia pathology in dorsal root ganglia organoid-derived sensory neurons

Abstract

Friedreich’s ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

A human stem cell-derived neurosensory-epithelial circuitry on-a-chip to model herpes simplex virus reactivation

“…Triple immunostaining identified peripheral sensory neurons expressing different combinations of TRK receptors confirming their correct differentiation in nociceptive (TRKA+/TRKC−), mechanoreceptive (TRKB+/TRKC+) and proprioceptive (TRKB−/TRKC+) neurons ( Figure S1D-F ). Bulk and single-cell RNA-seq confirmed and extended this analysis showing that DRGOs include also Satellite cells and Schwann cell progenitors, mirroring the overall cell composition of somatic DRGs (Mazzara et al 2020).…”

“…Interestingly, this similarity is not strikingly related to HSV-1 receptors, but is a more general feature of DRGOs, underlined from the close relation to primary DRG in regard Heparan sulfate biosynthetic pathways and, more in general, integrins ( Figure 2B-C ). Remarkably, such similarities between primary and stem cell derived ganglion neurons were evident even if DIV40 DRGO neurons are generally not completely mature yet (Mazzara et al 2020), and some differences are still present, such as for MAG , HS3ST3B1 and SCD1 ( Figure 2A) .…”

“…A thorough analysis of virus reactivation requires a humanized system where keratinocytes and neurons can be simultaneously cultured for extensive time developing direct connectivity. To this aim we decided to evaluate the possibility to develop a new neuron-keratinocyte co-culture system using hiPSC-derived dorsal root ganglia organoids (DRGOs) that we have recently derived and showed to generate molecularly-defined peripheral sensory neurons capable of making specific contacts with their respective target tissues (Mazzara et al 2020). DRGOs were obtained by differentiating transgene-free hiPSC-derived aggregates in 3D culture conditions and sequentially exposed to small molecules to induce peripheral nervous system identity ( Figure S1A ).…”

“…To confirm the DRGO sensory neurons competence for HSV infectivity, we next evaluated the main HSV cellular receptors expression in DRGOs. To this end, we carried out a computational analysis on global and single cell RNA-sequencing datasets on isolated DRGOs generated in our previous work (Mazzara et al 2020). When compared to iPSCs at global transcriptomic level, we confirmed that DIV40 DRGOs and primary DRGs are closely related with respect to the expression of the main genes involved in virus-cell contact, in particular the main HSV-1 receptor NECTIN1 , but also TNFRSF14 ( HVEM ), PILRA, NRP1 , MYH9 , ITGA5 , ITGA8 , ITGB6 and ITGB8 ( Figure 2A ).…”

“…Therefore, induction and reactivation from latency of herpesviruses on human DRGO through infection of primary keratinocytes sinaptically connected with them was not described yet. In this study, we took advantage of human iPSC-derived 3D dorsal root ganglion-like organoids (DRGOs), generated by in vitro differentiation of hiPSCs in 3D cultures (Mazzara et al 2020). Molecular and genomic analyses and topographic connectivity to peripheral targets have confirmed that DRGOs are populated by nociceptive, mechanoreceptive and proprioceptive neurons.…”