Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development

Abstract

Autonomic parasympathetic neurons (parasymNs) control unconscious body responses, including "rest-and-digest." ParasymN innervation is important for organ development, and parasymN dysfunction is a hallmark of autonomic neuropathy. However, parasymN function and dysfunction in humans are vastly understudied due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void as a versatile platform. Here, we developed a differentiation paradigm detailing the derivation of functional human parasymNs from Schwann cell progenitors. We employ these neurons (1) to assess human autonomic nervous system (ANS) development, (2) to model neuropathy in the genetic disorder familial dysautonomia (FD), (3) to show parasymN dysfunction during SARS-CoV-2 infection, (4) to model the autoimmune disease Sjögren's syndrome (SS), and (5) to show that parasymNs innervate white adipocytes (WATs) during development and promote WAT maturation. Our model system could become instrumental for future disease modeling and drug discovery studies, as well as for human developmental studies.

Keywords: COVID-19; Schwann cell progenitors; Sjögren’s syndrome; adipocyte innervation; autonomic nervous system; disease modeling; familial dysautonomia; human pluripotent stem cells; parasympathetic neurons; peripheral nervous system.

Figure 1.

SCP-derived neurons show parasympathetic identity. (A) Schematic illustration of the differentiation workflow to make symNs, SCs, and parasymNs from NCCs. (B) RT-qPCR analysis of day 30 parasymNs for parasymN markers. n=3–5 biological replicates. (C) RT-qPCR comparisons of SOX10 expression between SCs and parasymNs and ChAT/TH ratio between symNs and parasymNs on day 30. Student’s two-tailed t-test. n=3–6 biological replicates. (D) Western blot analysis of day 30 symNs and parasymNs. n=2 biological replicates. (E) Immunofluorescence images of hPSC-derived parasymNs for parasymN markers on day 30. (F) Quantification based on the immunofluorescence images. n=4–6 biological replicates. (G) PCA plot of parasymNs, symNs, SNs, MNs, and PFCs using bulk RNAseq. (H) Cartoon illustration shows the alignment of HOX genes in the parasympathetic and sympathetic nervous system. (I) RT-qPCR analysis of day 30 parasymNs for HOX genes. n=4 biological replicates. (J) Single cell transcriptomics defined the cellular composition in parasymN differentiation on day 30. Error bars=SEM. **p<0.01, ****P<0.0001. FC=fold change. See also Figure S1–S4.

Figure 2.

SCP-derived neurons display functional parasymN features. (A) Representative MEA heatmap of day 0 hPSCs and day 30 parasymNs. (B) ParaymN activity unstimulated or nicotine (1 μM) treated, measured by MEA. Student’s two-tailed t-test. n=4 biological replicates. (C) Acetylcholine (ACh) concentration in parasymN culture media measured by ELISA. n=3 biological replicates. (D) RT-qPCR of parasymNs for muscarinic receptors (MusRs). Student’s two-tailed t-test. Data of M2/4 were pooled and compared to M1/3/5. n=3–4 biological replicates. (E) ParaymN activity between control and BeCh (1 μM) or atropine (1 μM)-treated cells measured by MEA. Student’s two-tailed t-test. n=3 biological replicates. (F) Western blot analysis of 6-OHDA (100 μM) treated parasymN and symN 12 hr after treatment. n=3 biological replicates. (G) Changes of paraymN and symN electric activity over time after treatment with 6-OHDA. Values of parasymNs or symNs were normalized by their own vehicle treated groups, respectively (indicated by the red dotted line). Two-way ANOVA. n=3 biological replicates. (H) Immunofluorescence staining of 6-OHDA treated parasymN and symN 24 hr after treatment. n=4 biological replicates. (I) Top: Schematic illustration of hPSC-derived parasymNs and CMs co-culture. Bottom: Immunofluorescence images of the co-culture and the neural cardiac junction (NCJ, indicated by white arrows) highlighted in I’. (J) Cardiac action potential of the co-culture measured by MEA between control and nicotine (1 μM)-treated or blue light-treated cells. Student’s two-tailed t-test. n=6 for nicotine treatment and 4 for blue light biological replicates. Error bars=SEM. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001. FC=fold change. MFR=mean firing rate. See also Figure S5.

Figure 3.

Comparison of parasymN and symN development. (A) Cell migration of day 16 SCP and day 14 symNblast was compared by plating down spheroids and assessing neuron migration out of the spheroid after 24 hr. n=4 biological replicates. (B) Left: Representative immunofluorescent images of neurons after day 14 symNblasts were differentiated in the respective symN or parasymN medium. Top right: Quantification of neural numbers (TH⁺ for symNs, ChAT⁺ for parasymNs) from both media. Unpaired Student’s t-test. n=3 biological replicates. Bottom right: Comparison of HMX3 mRNA level between SCP- or symNblast-derived neurons in parsymN medium. Unpaired Student’s t-test. n=3 biological replicates. (C) Sample distance plot of day 16 SCP and day 14 symNblast by bulk RNAseq. (D) PCA plot of day 16 SCP and day 14 symNblast by bulk RNAseq. (E) Pathway enrichment analysis showed GO terms upregulated in day 14 symNblast compared to day 16 SCP. (F) PCA plot of day 10 NC, day 16 SCP, and day 30 parsymN. (G) Heatmap showed expression profile of parasymN and symN developmental signatures in day 16 SCP and day 30 parsymN. (H) Protein-protein interaction analysis of upregulated genes in parasymN and symN differentiations. (I) Representative immunofluorescent images and mRNA levels of CD274 in parasymNs and symNs. Unpaired Student’s t-test. n=3–4 biological replicates. *p<0.05, **p<0.01.

Figure 4.

Parasympathetic hyperactivity and impaired autonomic crosstalk in FD. (A) Inset: Immunofluorescence images of control and FD parasymNs for PRPH. MEA analysis of control and FD parasymNs. Two-way ANOVA. n=3–5 biological replicates. (B) Fold changes of neural activity of FD symNs and parasymNs relative to control symNs and parasymNs. Student’s two-tailed t-test. n=4–11 biological replicates. (C) RT-qPCR analysis for ELP1 splicing in ctrl and FD parasymNs. n=4 biological replicates. (D) Cartoon illustration of the effects of ACh released by parasymNs to CMs and SMCs. (E) Muscular activities of hPSC-CMs and hPSC-SMCs co-cultured with control or FD parasymNs. Student’s two-tailed t-test. n=4 biological replicates. (F) RT-qPCR of control and FD parasymNs for signal transduction markers. Multiple unpaired Student’s t-test. n=3–5 biological replicates. (G) Sample distance plot of ctrl and FD parasymNs by bulk RNAseq. (H) PCA plot of day ctrl and FD parasymNs by bulk RNAseq. (I) Pathway enrichment analysis showed GO terms upregulated in FD parasymNs compared to ctrl. (J) Left: Schematic illustration of conditional medium treatments. Right: MEA analysis of FD symNs and parasymNs treated with conditional media from control and FD symNs and parasymNs. One-way ANOVA. n=5–9 biological replicates. (K) Cartoon illustration shows that in FD PNS, both PSNS and SNS are hyperactive, but SNS hyperactivity is stronger. It also shows that the crosstalk between PSNS and SNS is impaired. Error bars=SEM. *p<0.05, **p<0.01, ****P<0.0001. FC=fold change. MFR=mean firing rate. See also Figure S6–S7.

Figure 5.

hPSC-derived parasymNs in COVID-19 mimicking studies. (A) Cartoon illustration of how SARS-CoV-2 infection leads to an imbalanced RAAS, which leads to an imbalanced ANS that worsens the cardiovascular complications observed in COVID-19 patients. (B) RT-qPCR of parasymNs for ACE2 and AGTR1/2. n=3 biological replicates. (C) Representative immunofluorescence images of parasymNs for AGTR1/2. (D) Schematic illustration of SARS-CoV-2 infection. (E) Representative immunofluorescence images of neurons after 72 hrs infection with WA1 SARS-CoV-2. (F) MEA analysis comparing neural activity of control and AngII-treated parasymNs. Student’s two-tailed t-test. n=10 biological replicates. (G) Schematic illustration of the potential anti-inflammatory effect of parasymN conditional media in an anti-inflammation assay. (H) ROS level of hPSC-derived CMs with each treatment was shown as CM-H2DCFDA intensity measured by ELISA. One-way ANOVA. n=4 biological replicates. (I) Cardiac activity of hPSC-derived CMs with each treatment measured by MEA. One-way ANOVA. n=3 biological replicates. (J) Beating variability of hPSC-derived CMs with each treatment measured by MEA. One-way ANOVA. n=3 biological replicates. Error bars=SEM. *p<0.05, **p<0.01, ***P<0.001. FC=fold change. MFR=mean firing rate.

Figure 6.

hPSC-parasymN model of Sjögren’s syndrome (SS). (A) Schematic illustration of the antibody-based complement-dependent cytotoxicity assay. (B) Immunofluorescence images using anti-human IgG secondary antibody showed SS IgG antibodies attached to parasymN clusters. Comparisons of parasymNs treated with or ctrl or SS IgG are: (C) Quantification of positive cell numbers in neural clusters. Three clusters in each biological replicate were analyzed from 3–4 biological replicates. One-way ANOVA. (D) MEA analysis of parasymNs. One-way ANOVA. n=4 biological replicates. (E) ACh release in parasymN cultures. One-way ANOVA. n=3 biological replicates. (F) AChE activity from parasymNs. One-way ANOVA. n=4 biological replicates. (G) ROS level of parasymNs was compared using CM-H2DCFDA. One-way ANOVA. n=3 biological replicates. (H) Percentage of changes in Ca²⁺ dynamics in salivary cells upon nicotine stimulation and SS patient IgG treatments in the co-culture compared to control IgG. n=4 biological replicates. (I) Cartoon illustration summarizing that in SS patients, parasymNs can be targeted by auto-Abs, which decreases parasymN activity. ACh extracellular level is increased, and AChE is reduced, possibly due to the autoimmune response. Error bars=SEM. *p<0.05, **p<0.01, ***p<0.001, ****P<0.0001. FC=fold change. MFR=mean firing rate. See also Figure S8–S9.

Figure 7.

hPSC-derived parasymNs target white adipocytes in vitro. (A) Schematic illustration of human parasymN and mouse WAT co-culture. (B) Immunofluorescence images of the co-culture. Comparisons of WAT with or without parasymNs are: (C) RT-qPCR analysis of WAT using human VACHT primer. Data was shown in Ct value. The Ct for of the detection limit is 40. n=5 biological replicates. (D) Left: Immunofluorescence images compare FABP4 expression in the adipocytes. Images were taken at the same exposure. Right: Image quantification of FABP4 staining. Student’s two-tailed t-test. n=6 biological replicates. (E) Left: Fluorescence images compare adipogenicity in the adipocytes using LipidSpot staining. Right: Image quantification of LipidSpot intensity. Student’s two-tailed t-test. n=4 biological replicates. (F) Left: Immunofluorescence images for Tuj1, FABP4, and DAPI in adipocyte cultures. Right: Quantification of nucleus size. 10 nuclei in each biological replicate were analyzed from 3 biological replicates. Student’s two-tailed t-test. (G) Glucose concentration in the culture media, as well as glucose uptake (intracellular) level in WAT. Student’s two-tailed t-test. n=4. (H) Glycerol release from adipocytes representing the lipolytic activity measured by ELISA. 1 μM nicotine was applied to activate parasymNs. Multiple unpaired Student’s t-test. n=4–6 biological replicates. (I) Glycerol release upon treatment with 10 μM isoproterenol. One-way ANOVA. n=4 biological replicates. (J) RT-qPCR for lipolytic markers using mouse primers in co-culture. Multiple unpaired Student’s t-test. n=4 biological replicates. Error bars=SEM. *p<0.05, **p<0.01, ****P<0.0001. FC=fold change. See also Figure S10.