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. 2020 May 8;10(1):7752.
doi: 10.1038/s41598-020-64831-6.

Primary Proprioceptive Neurons From Human Induced Pluripotent Stem Cells: A Cell Model for Afferent Ataxias

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Free PMC article

Primary Proprioceptive Neurons From Human Induced Pluripotent Stem Cells: A Cell Model for Afferent Ataxias

Chiara Dionisi et al. Sci Rep. .
Free PMC article

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.

Conflict of interest statement

M.P. has received compensation as a consultant and member of the scientific advisory board of Voyager Therapeutics and owns stock in the company. He also has consulted for Apopharma and Biomarin and received compensation. M.P. is a member of the Scientific Committee of the Italian Telethon. C.D., M.R., M.C, and S.N.S. declare no potential conflict of interest.

Figures

Figure 1
Figure 1
Scheme of the differentiation protocol.
Figure 2
Figure 2
Time course of the expression (log2 scale) of nine transcription factors during iPSC differentiation to sensory neurons in two control and three FRDA lines. mRNA levels were determined by quantitative RT-PCR in duplicate experiments and normalized to levels in control iPSCs.
Figure 3
Figure 3
(A–C) SOX10 (green) and BRN3A (red) expression by IF in tubular aggregates of neural precursors at day 5. Nuclei are shown in blue (Hoechst). (D–F) BRN3A (green) TUBB3 (red) expression by IF in differentiated sensory neurons at day 15. Nuclei are shown in blue (Hoechst).
Figure 4
Figure 4
Time course of the expression (log2 scale) of Trk receptors, p75, vGLUT1, PV, S100 and FXN during iPSC differentiation to sensory neurons in two control and three FRDA lines. mRNA levels were determined by quantitative RT-PCR in duplicate experiments and normalized to levels in control iPSCs.
Figure 5
Figure 5
(A,E) TrkA; (B,F) TrkB; (C,G) TrkC; (D,H) PV expression by IF in differentiated sensory neurons after 7 days of exposure to neurotrophic factors (day 15 of differentiation). Staining for all antibodies is in green. Nuclei are shown in blue (Hoechst).
Figure 6
Figure 6
Bar graph showing the percentage of TrkA+, TrkB+, TrkC+ and PV+ cells by quantification of IF images of cultures after 7 days of exposure to neurotrophic factors (day 15 of differentiation).
Figure 7
Figure 7
(A) Expression of Trk receptors assessed using flow cytometry on cultures after 7 days of exposure to neurotrophic factors (day 15 of differentiation). Unstained neurons were used as negative control (light blue histograms). In each panel, the average of positive cells observed for each marker in all trials performed (n = 4) is reported: TrkA+ neurons represented the 1–2% of differentiated cultures (violet histogram), TrkB+ the 25–20% (green histogram), while TrkC+ cells were the 60–70% (red histogram) in analyzed samples. Channels used for detection included: DAPI (Alexa Fluor 405), FITC (Alexa Fluor 488), PE (Phycoerythrin). (B) Fold-difference of Trk receptor and proprioceptor marker expression by quantitative RT-PCR analysis in sorted cells vs. starting sensory neurons cultures.
Figure 8
Figure 8
(A) (Up) Representative image of biocytin-filled TrkC+ neuron. (Down) Representative image of neurons identified with a 63x water immersion objective and an infrared CCD camera during electrophysiological recording. They showed the typical morphology of large sensory pseudo-unipolar neurons. Time course analysis of electrophysiological excitability of differentiated neurons. (B) Representative traces of current-clamp recordings in cells at day 7 (left) 10 (middle) or 12 (right) of treatment with neurotrophic factors (days 15, 18, 20 of differentiation), in response to depolarizing current injection steps at 0 pA (lower trace) 20, 30 and 100 pA (higher trace). (C) Representative traces of current-clamp recordings in cells at day 12 of treatment with neurotrophic factors, in response to 0 (lower traces) 20, 30 and 100 pA (higher traces) of depolarizing current injection steps. Three different responses were observed: regular APs firing with firing frequency increasing for higher current injections (left); spontaneous activity with rapid accommodation following increasing step current injections (middle); single AP in response to higher injecting currents (right).

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