Satellite glial contact enhances differentiation and maturation of human iPSC-derived sensory neurons

Abstract

Sensory neurons generated from induced pluripotent stem cells (idSNs) are used to model human peripheral neuropathies; however, current differentiation protocols produce cells with an embryonic phenotype. Peripheral glia contact sensory neurons early in development and contribute to formation of the canonical pseudounipolar morphology, but these signals are not encompassed in current idSN differentiation protocols. Here, we show that terminal differentiation of idSNs in coculture with rat dorsal root ganglion (rDRG) satellite glia and glial precursors (rSG) advances differentiation and maturation. Cocultured idSNs develop pseudounipolar morphology through contact with rSG. In addition to morphological changes, idSNs terminally differentiated in coculture exhibit enhanced action potential firing, more mature gene expression, and increased susceptibility to paclitaxel-induced axonal degeneration. Thus, idSNs differentiated in coculture with rSG provide a better model for investigating human peripheral neuropathies.

Keywords: CIPN; DRG; idSN; neuropathy; paclitaxel; pseudounipolar; satellite glia; sensory neuron.

Figure 1

Human iPSC-derived sensory neurons (idSNs) differentiated in coculture with embryonic rat DRG (rDRG) glia exhibit mature morphology and function (A) Coculture protocol; (B) idSN morphologies. TUJ1 = green, human nuclei = red, DAPI = cyan. Scale bars, 25 μm. PU = pseudounipolar. PU-AB: white arrowhead indicates accessory branch. (C) Frequency of morphologies n = 4 bio rep, 2 iPSC lines. (D) Measurement of axonal diameter in PU cells. n = 76 cells, 3 bio rep, 2 iPSC lines. (E) Raster plots over 2.5 min. Black lines = spikes, blue lines = bursts. (F) Average # active electrodes when average was ≥5. Coculture n = 51 wells, idSN alone n = 28 wells, Glia alone n = 24 wells, n = 6 bio rep. 3/iPSC line. (G–I) # of spikes (F), mean firing rate (G), and # of bursts (H) when average # active electrodes were ≥5. Coculture n = 49 wells, idSN alone n = 27. p < 0.05 = significant. Related to Figure S1.

Figure 2

iPSC-derived neural crest precursors (idNCPs) terminally differentiated in coculture are more advanced in neuronal differentiation (A) Differential gene expression (DGE) in coculture vs. alone. (B) Gene Ontology (GO) terms upregulated in coculture vs. idSNs alone, related to neuronal development. (C) GO terms downregulated in coculture vs. idSNs alone, related to neuronal development. Coculture n = 3, idSN alone n = 4. p adj.<0.05. (D) PRPH and SOX10 expression. PRPH = magenta, SOX10 = green, DAPI = cyan, human nuclei = red (coculture only). Scale bars, 50 μm. (E and F) (E) Particle analysis of PRPH, coculture n = 48 fields, idSN alone n = 47 fields (F) % SOX10+ human, coculture n = 48 fields, idSN alone n = 48 fields, 3 bio rep, 2 iPSC lines. p < 0.05 = significant. Related to Figure S2.

Figure 3

Neuronal maturation media (NMM) enriches for satellite glia (SG) (A–F) (A) DGE, NMM-treated rDRG glia vs. rDRG glia in glia base media. (B and C) GO terms upregulated (B) and downregulated (C) in NMM-treated rDRG glia vs. rDRG glia in glia base media. (D) DGE, cocultured rDRG glia vs. rDRG glia maintained in glia base media. (E and F) GO analysis terms upregulated (E) and downregulated (F) in cocultured rDRG glia vs. rDRG glia in glia base media. (G) DGE, cocultured rDRG glia vs. NMM-treated rDRG glia. n = 3–4/group, 2 iPSC lines p adj.<0.05. Related to Figure S3.

Figure 4

Rat satellite glia (rSG) are responsible for advanced maturation of idSNs (A) rDRG glia in glia base media 2 weeks or treated with NMM days 7–14. S100b = green, GFAP = red (top) p75 = red (bottom), DAPI = cyan. Scale bars, 50 μm, arrowhead = large immature glia, arrows = small mature rSG. (B) Top: glia in base media 2 weeks or treated with NMM days 7–14. Bottom: cocultured idSNs and idSNs alone. S100b = magenta (top), TUJ1 = magenta (bottom), MBP = green, DAPI = cyan. Scale bars, 50 μm. (C) Cocultured idSNs (TUJ1 = green, human nuclei = red, p75 = magenta). DAPI = cyan. Scale bars, 50 μm. (D) #p75+ cell contacts on idSNs in coculture from Figure 1C. One-way ANOVA, p < 0.05 = significant. (E) Image stack illustrating rSG contact with PU cell, 0.75 μM between optical sections. TUJ1 = green, human nuclei = red, p75 = magenta, DAPI = cyan. Arrow shows contact between p75+ processes and PU axon stem/cell body. Scale bars, 25 μm. (F) FN1 reads normalized to Actb, from Figure 3 RNA-seq. (G) idSNs in 3T3 coculture and alone at 20× (top) and 40× (bottom). TUJ1 = green, human nuclei = red (coculture only), DAPI = blue. Scale bars,100 μm (top) and 50 μm (bottom). (H) Morphology frequencies of idSNs in 3T3 coculture and alone. n = 3 bio rep, 2 iPSC lines. Related to Figure S3.

Figure 5

rSG-secreted factors do not induce pseudounipolar morphology in idSNs (A) Transwell coculture protocol. (B) idSNs cultured in transwell coculture or alone. TUJ1 = green, BRN3A = red, DAPI = blue. Scale bars, 50 μm. (C) Morphology frequencies in coculture and alone. n = 3 bio rep, 1 iPSC line (D) Protocol for idSN treatment with coculture spent media. (E) idSNs treated with coculture spent media or untreated. TUJ1 = green, BRN3A = red, DAPI = blue. Scale bars, 50 μm. (F) Morphology frequencies in coculture-media-treated and untreated idSNs, n = 3 bio rep, 2 iPSC lines.

Figure 6

Physical contact between rSG and idSNs is essential for pseudounipolarization (A) Protocol for idSN coculture with methanol fixed rSG. (B) idSNs in coculture with methanol fixed rSG, normal rSG, or alone. TUJ1 = green, p75 = magenta, human nuclei = red, DAPI = cyan. idSNs alone were not stained for human nuclei. Scale bars, 50 μm. (C) Morphology frequencies in cocultured idSNs with methanol fixed rSG, normal rSG, and alone. n = 4 biological replicates, 2 iPSC lines. (D) Protocol for idSN coculture with SEMA6a and GFP knockdown in rSG. (E) idSNs in coculture with SEMA6a knockdown rSG or GFP knockdown rSG. SEMA6a = magenta, TUJ1 = red, GFP = green, DAPI = cyan. Scale bars, 50 μm overall and 10 μm on inset. (F) Morphology frequencies in idSNs coculture with SEMA6a knockdown and GFP knockdown n = 3 bio rep, 2 iPSC Lines. (G) Integrated density particle analysis of SEMA6a, n = 19 fields and sh-GFP images n = 17 fields, 2–3 bio rep, 2 iPSC lines. Student’s t test, p < 0.05 = significant. Related to Figure S4.

Figure 7

Cocultured idSNs are more susceptible to paclitaxel-mediated neurodegeneration (A and C) (A) Deng et al., (2023) generated idSNs and (C) RealDRG idSNs in coculture or alone, treated with DMSO or 60 and 600nm paclitaxel, respectively, 48h. TUJ1 = green, GJA1 = magenta, human nuclei = red (coculture only), DAPI = cyan. idSNs alone were not stained for human nuclei. Scale bars, 50 μm. (B and D) Percent area TUJ1. Two-Way ANOVA with Bonferroni post-hoc, p < 0.05 = significant, p < 0.0001 = ^∗∗∗∗. n = 64 fields, 4 biological replicates. (E) NMM-treated rSG (days 7–14), treated with 0.2% DMSO or 60 nM paclitaxel 48 h. S100b = red, Ki67 = green (top), cleaved caspase-3 = green (bottom), DAPI = blue. Scale bars, 50 μm. (F) Total # nuclei in fields stained for ki67. (G) % nuclei + for ki67. (F–G) DMSO n = 48 fields, Pac. n = 47 fields, 3 bio rep. (H) Total # nuclei in fields stained for caspase3. (I) % caspase3+ nuclei. (H and I) DMSO n = 48 fields, Pac. n = 48 fields. 3 bio reps. (F–I) Student’s t test p < 0.05. (J) idSNs treated with chemotherapy-conditioned media from cocultures, idSNs alone, and rSG alone. TUJ1 = green, DAPI = blue. Scale bars, 50 μm. (K) Percent area TUJ1 coculture treatment n = 45, idSN alone treatment n = 41, glia-alone treatment n = 48 fields, 3 bio rep. One way-ANOVA, Bonferroni post-hoc, p < 0.05 = significant. Related to Figure S5.