Induced pluripotent stem cell-derived primary proprioceptive neurons as Friedreich ataxia cell model

Abstract

Human induced pluripotent stem cells (iPSCs) are used to generate models of human diseases that recapitulate the pathogenic process as it occurs in affected cells. Many differentiated cell types can currently be obtained from iPSCs, but no validated protocol is yet available to specifically generate primary proprioceptive neurons. Proprioceptors are affected in a number of genetic and acquired diseases, including Friedreich ataxia (FRDA). FRDA is a recessive neurodegenerative and systemic disease due to epigenetic suppression of frataxin (FXN) expression caused by the presence of expanded GAA repeats at the FXN locus. The most characteristic early neuropathologic finding in FRDA is the loss of large primary proprioceptive neurons in the dorsal root ganglia (DRGs), with associated loss of large myelinated fibers in the dorsal roots and in the posterior columns of the spinal cord. Both a developmental deficit and progressive neurodegeneration are thought to underlie the loss of proprioceptors in FRDA, though the relative contribution of these two components is unclear. The basis of the high specific vulnerability of proprioceptors in FRDA is also unknown. In order to address these open questions about FRDA pathogenesis and at the same time develop a cell model that can be applied to other conditions primarily affectingproprioceptors, we set up a protocol to differentiate iPSCs into primary proprioceptive neurons. We modified the dual-SMAD inhibition/WNT activation protocol, previously used to generate nociceptor-enriched cultures of primary sensory neurons from iPSCs, to favor instead the generation of proprioceptors. We succeeded in substantially enriching iPSCderived primary sensory neuron cultures in proprioceptors, largely exceeding the proportion normally represented by these cells in dorsal root ganglia. We also showed that almost pure populations of proprioceptors can be purified from these cultures by fluorescence-activated cell sorting. Finally, we demonstrated that iPSCs from a FRDA patient can generate normal appearing proprioceptors but have subtle differentiation deficits and more limited survival. Corresponding authorCorrespondence to Massimo Pandolfo.

Perspectives on current models of Friedreich’s ataxia

“…FRDA iPSC-based neurons exhibited reduced mitochondrial transcripts and altered expression levels in transcripts involved in ECM organization and focal adhesion (Lai et al, 2019). Primary proprioceptive neurons differentiated from FRDA iPSCs had reduced expression of proprioceptor-specific markers and exhibited shorter survival in vitro (Dionisi et al, 2020).…”

“…FXN downregulation observed in fibroblasts was found to be retained in the iPSCs (Ku et al, 2010). Since then, several additional FRDA patient-derived iPSCs were generated to study the pathophysiology of this disease (Liu et al, 2011;Bolotta et al, 2019;Schreiber et al, 2019;Dionisi et al, 2020;Mazzara et al, 2020;Angulo et al, 2021;Kelekci et al, 2021).…”

Isolation of human pluripotent stem cell-derived sensory neuron subtypes by immunopanning

“…The human pluripotent stem cell (hPSC) technology can close this gap. Indeed, many protocols have been established to differentiate hPSCs into SNs, focusing on nociceptors (Pomp et al, 2005;Chambers et al, 2012;Young et al, 2014;Boisvert et al, 2015;Wilson et al, 2018;Holzer et al, 2022), mechanoreceptors (Schrenk-Siemens et al, 2015;Nickolls et al, 2020;Zhu et al, 2021), or proprioceptors (Dionisi et al, 2020). Other protocols can generate all SN subtypes in bulk DRG-like cultures (Alshawaf et al, 2018;Saito-Diaz et al, 2021).…”