Primary proprioceptive neurons from human induced pluripotent stem cells: a cell model for afferent ataxias

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). To develop a cell model that can be applied to conditions primarily affecting proprioceptors, 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 iPSC-derived primary sensory neuron cultures for proprioceptors, up to 50% of finally differentiated neurons, largely exceeding the proportion of 7.5% 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 the protocol can be used to generate proprioceptors from iPSCs from FRDA patients, providing a cell model for this genetic sensory neuronopathy.

Derivation of Peripheral Nociceptive, Mechanoreceptive, and Proprioceptive Sensory Neurons from the same Culture of Human Pluripotent Stem Cells

“…This was confirmed by low expression of markers of autonomic ( ASCL1 ), motor ( HB9 and OLIG2 ), CNS ( TBR1 ), and enteric neurons ( EDNRB ) ( Figures S1 I and S1J). However, we found that the remaining 30%–40% of the cells in the cultures have a character of myofibroblasts (α-smooth muscle actin, desmin) and Schwann cells ( MPZ , MBP ) ( Figures S1 K and S1J), similar to previous reports ( Dionisi et al., 2020 ).…”

“…Lastly, a recent study showed a method to generate proprioceptors (70% of SNs) from SOX10 + NCCs using high concentrations of NT3 and low concentrations of NGF, GDNF, and BDNF ( Dionisi et al., 2020 ). The authors also see increases of NGN1 and NGN2 expression.…”

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

“…It is reported that 2D iPSC-SNs form unorganized clusters in a random pattern with unproven concomitant participation of different neuron subtypes and glial cells, fail to recapitulate the DRGs spatial architecture and cellular diversity 35 , 36 . Of note, even if some recent studies showed new derivation of FRDA SNs by direct cell reprogramming or iPSC differentiation 37 , 38 , when we tried to generate SNs from FRDA patient iPSCs, an extensive cell death occurred which prevented obtaining neuronal cultures with sufficient numbers of PRPH + /BRN3A + sensory neurons (Supplementary Fig. 3c ).…”

Structural and Biological Basis for Proprioception

“…Accordingly, typical clinical consequences of pathology affecting proprioceptive neurons, in addition to afferent ataxia, include loss of deep tendon (stretch) reflexes and of conscious perception of position and movement of body parts, often associated with loss of perception of vibration [9]. Recently, Dionisi and co-workers have obtained proprioceptive primary sensory neurons from iPSCs, and the generation of intrafusal fibers in vitro are opening new perspectives for the treatment of some ataxia linked to altered primary proprioceptive neurons [100,101].…”