Differentiation and on axon-guidance chip culture of human pluripotent stem cell-derived peripheral cholinergic neurons for airway neurobiology studies

Abstract

Airway cholinergic nerves play a key role in airway physiology and disease. In asthma and other diseases of the respiratory tract, airway cholinergic neurons undergo plasticity and contribute to airway hyperresponsiveness and mucus secretion. We currently lack human in vitro models for airway cholinergic neurons. Here, we aimed to develop a human in vitro model for peripheral cholinergic neurons using human pluripotent stem cell (hPSC) technology. hPSCs were differentiated towards vagal neural crest precursors and subsequently directed towards functional airway cholinergic neurons using the neurotrophin brain-derived neurotrophic factor (BDNF). Cholinergic neurons were characterized by ChAT and VAChT expression, and responded to chemical stimulation with changes in Ca²⁺ mobilization. To culture these cells, allowing axonal separation from the neuronal cell bodies, a two-compartment PDMS microfluidic chip was subsequently fabricated. The two compartments were connected via microchannels to enable axonal outgrowth. On-chip cell culture did not compromise phenotypical characteristics of the cells compared to standard culture plates. When the hPSC-derived peripheral cholinergic neurons were cultured in the chip, axonal outgrowth was visible, while the somal bodies of the neurons were confined to their compartment. Neurons formed contacts with airway smooth muscle cells cultured in the axonal compartment. The microfluidic chip developed in this study represents a human in vitro platform to model neuro-effector interactions in the airways that may be used for mechanistic studies into neuroplasticity in asthma and other lung diseases.

Keywords: asthma; cholinergic; neuron; organ-on-chip; stem cell.

FIGURE 1

Derivation of vagal NCCs from hPSCs. (A) Schematic overview of vagal NCC induction from hPSCs. H9WA09 cells were directed into a vagal NCC faith using dual SMAD inhibition, and early WNT activation, r. a. Was added to direct NCCs from a cranial into a more distal phenotype. Knock-out serum replacer medium (KSR) was gradually replaced by N2 medium. Vagal NCCs were cultured in spheroids for 4 days to complete maturation. Created with BioRender.com. (B) Bright images of the differentiating cells over time. Scale bar = 200 µm. (C) OCT4 expression of Day 0 pluripotent cells. Scale bar = 50 µm. (D) SOX10 expression of Day 16 vagal NCC spheroids. Scale bar = 50 µm. (E,F) Gene expression of several markers for pluripotency, NCC, and vagal NCC. After 12 days, a decrease in the pluripotency genes OCT4 and Nanog was observed. The NCC marker PAX3 was upregulated, in combination with the vagal NCC markers HOXB3 and HOXB5 (N = 13) (G,H). FACS analysis showed that 90.48% (±2.83%) of the vagal NCCs were p75+-HNK1+ double positive. (N = 12) A paired t-test was performed to calculate statistical significance between means. ***p < 0.001; ****p < 0.0001 compared to day 0.

FIGURE 2

Differentiation of peripheral cholinergic neurons. (A) Schematic overview of hPSC differentiation into peripheral cholinergic neurons. Created with BioRender.com. (B) Brightfield images of the differentiating cells over time. (C–E) Immunofluorescence images of differentiating cells over time, showing the development of β-3-tubulin expression. Over time a greater neuronal network is being formed. Day 17, scale bar = 20 µm. Day 35 and day 46, scale bar = 100 µm. (F–L) Gene expression of airway cholinergic neuronal development over time. Gene expression was examined at different time points: Day 16, day 25, day 35, and day 50. ASCL1 (F) and phoxb2 (G) are essential for neuronal development and increased over the course of day 50. The pan-neuronal markers PGP9.5 (H) and β-3-tubulin (I) indicate the formation of neurons, in combination with peripherin (J) they indicate the development of peripheral neurons. VAChT (K), indicating the formation of cholinergic neurons, was higher expressed over the course of 50 days. TH (L) showed a peak at day 35 before declining in expression towards day 50. TrkB (M) increased in expression over the course of 50 days (N = 8) A mixed-effect analysis followed by Tukey’s multiple comparisons test was performed to calculate statistical significance between means. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, compared to day 16.

FIGURE 3

Peripheral cholinergic neurons (A–D). Immunofluorescence images characterizing neurons. β-3-tubulin or PGP9.5 were used as a pan-neuronal marker. The neurons show the presence of VAChT (A) after 50 days of differentiation, in addition to SYP (B) and peripherin (C). A smaller proportion of the neurons show TH⁺ after 50 days. (E–G) FACS analysis demonstrated that 27.7% (±3.8%) of the neurons are β-3-tubulin⁺-ChAT⁺, indicative of cholinergic neurons. A representative FACS analysis plot is shown in E, panel F shows the quantification (n = 8). (G) Quantification of IF staining of β-3-tubulin (n = 6), VAChT (n = 8), and TH (n = 8) showed that the majority of sprouting cells expressed β-3-tubulin and VAChT, whilst TH was only sparsely present. (H) PCA analysis of RNAseq data showed that hPSC-derived airway neurons were distinct from airway epithelial cells or fibroblasts, but showed more overlap with hPSC-derived peripheral neurons or brain cholinergic neurons. (I) Despite similarities with brain cholinergic neurons in the PCA analysis, peripheral cholinergic neurons showed a distinct expression of central and peripheral markers with the central markers ISL1, ISL2, OLIG2 and NEUROG1 being enriched in brain cholinergic neurons and the peripheral marker PRPH being enriched in peripheral cholinergic neurons. Figures were presented as mean ± SEM.

FIGURE 4

hPSC-derived peripheral cholinergic neurons on a chip. (A) Timeline of differentiation of peripheral neurons from hPSCs via vagal NCCs. In 50 days, peripheral cholinergic neurons were generated in the presence of BDNF. (B) If images of different stages of differentiation of hPSCs. Left: OCT4⁺ hPSC colony at day 0 of differentiation. Scalebar = 200 µm. Middle: SOX10⁺ vagal NCC spheroids at day 16 of differentiation. Scalebar = 200 µm. Right: β-3-tubulin⁺ airway neurons at day 50 of differentiation. Scalebar = 50 µm. (C) Schematic overview of the timeline of stemcell seeding into the chips after fabrication; the chips were first coated with PLO/LM/FB, after which vagal NCC spheroids were dissociated and seeded into the chip. The medium was changed 2-3 times per week. Axons started to grow out of the microchannels into the axonal compartment after 6 days. Neurons were cultured into the chip up to day 50. (D–F): IF images of β-3-tubilin⁺ neurons showing increased axonal outgrowth over time. Scalebar = 500 µm (D). At day 23 of differentiation (7 days on chip) some axonal outgrowth was visible, which increased towards day 35 (E) and day 50 (F). (G,H) IF images of peripheral neurons showing the majority of neurons was VACHT⁺ (G, scalebar = 200 µm) and peripherin⁺ (H, scalebar = 50 µm). (I–N). Schematic overview of Ca²⁺ imaging of hPSC-derived neurons cultured in six wells-plates or on-chip, plus traces of Ca²⁺ response of the generated neurons. KCl (60 mM) was added to the neurons or to a medium reservoir of the somal compartment. The time point of adding KCl is indicated with a black arrow. I-J. KCl stimulation and Ca²⁺ response of generated neurons cultured in a six wells-plate. (K–L) KCl stimulation and Ca²⁺ response of generated neurons cultured on-chip, neurons and axons were imaged in the somal compartment. (M,N) KCl stimulation and Ca²⁺ response of generated neurons cultured on-chip, axons were imaged in the axonal compartment. Shown are five representative traces for each experimental design, taken from n = 5 differentiations, each performed in 1-3 separate chips. Created with BioRender.com.

FIGURE 5

Comparing cell culture on-chip and in conventional cell culture. (A) Schematic overview of co-culture of neurons and ASM cells in the chip. In the left main compartment (blue) of the chip, neurons were seeded and in the right main compartment (red) ASM cells were seeded. Axons connected the two main compartments by growing for the neuronal to the axonal compartment. To compare, cells were cultured in a 6WP. Created with BioRender.com. (B) Left Brightfield images of neuronal-like SH-SYSY cells cultured both in a 6WP and in a chip. Right: Brightfield images of ASM cells cultured both in a 6WP and in a chip. Both SH-SY5Y and ASM cells had a similar morphology independent of the culture vessel. (C) IF image of an ASM-neuronal co-culture on-chip showed that both cell types are confined to their own compartment. Red: β-3-tubulin; green: Phalloidin. Scalebar = 200 µm. (D,E) mRNA expression of ASM cells cultured both in the chip and in a 6WP. The expression of ACTA2 and the CHRM3 receptor are similar for both culture vessels. F-G mRNA expression of cells isolated from individual channels. (F) ACTA2 expression of cells from either the somal compartment or the axonal compartment showed that ACTA2 was mainly expressed in the axonal compartment. (G) TUBB3 expression of cells isolated from either the somal or the axonal compartment showed that TUBB3 was mainly expressed in the somal compartment. (H,I) Live cell Ca²⁺ imaging using Fluo-4. MCh (10 µM) stimulation of ASM showed a similar trace for ASM both cultured in a 6WP or on-chip. The time point of adding MCh is indicated with a black arrow. Shown are individual traces of 10 regions of interest taken from n = 6 chips.

FIGURE 6

Co-cultures of hPSC-derived airway neurons and ASM cells on a chip (A) Schematic overview of the stem cell and ASM cell seeding into the chips after fabrication; Vagal NCCs were seeded and cultured in PLO/LM/FB coated chips up to day 45. The medium was changed 2-3 times per week. At day 45, ASM cells were seeded in the axonal compartment. Both cells were co-cultured for 5 days, up to day 50. (B) IF image of co-culture showing the expression of PGP9.5 and SYP in generated neurons. Scalebar = 50 µm. (C) IF image ASM cells and hPSC derived neurons. Phalloidin captured the actin-rich ASM cells and also the neurons (green). β-3-tubilin⁺ neurons innervate ASM in the axonal compartment (red). Scalebar = 200 µm. (D) Close-up of IF image of co-culture. White arrows indicate some innervating neurons (red). Scalebar = 100 µm. Created with BioRender.com.