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Differentiating visceral sensory ganglion organoids from induced pluripotent stem cells

Nature Methods volume 21, pages 2135–2146 (2024)Cite this article

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Abstract

The ability to generate visceral sensory neurons (VSN) from induced pluripotent stem (iPS) cells may help to gain insights into how the gut–nerve–brain axis is involved in neurological disorders. We established a protocol to differentiate human iPS-cell-derived visceral sensory ganglion organoids (VSGOs). VSGOs exhibit canonical VSN markers, and single-cell RNA sequencing revealed heterogenous molecular signatures and developmental trajectories of VSGOs aligned with native VSN. We integrated VSGOs with human colon organoids on a microfluidic device and applied this axis-on-a-chip model to Alzheimer’s disease. Our results suggest that VSN could be a potential mediator for propagating gut-derived amyloid and tau to the brain in an APOE4- and LRP1-dependent manner. Furthermore, our approach was extended to include patient-derived iPS cells, which demonstrated a strong correlation with clinical data.

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Fig. 1: Differentiating OEPD and EpP from iPS cells.
Fig. 2: Canonical VSN marker expression in VSGOs differentiated from EpP.
Fig. 3: VSGO scRNA analysis.
Fig. 4: Establishment of 3D axis-on-a-chip connecting VSGO and HCO using a microfluidic chip.
Fig. 5: Diverse assays using VSGOs for comparing APOE3/4 lines.
Fig. 6: Aβ and tau propagation assay of patient-derived VSGOs.

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Data availability

The human reference genome (GRCh38-2024-A) for the scRNA analysis was provided by 10x Genomics and is available at https://www.10xgenomics.com/support/software/cell-ranger/downloads. The scRNA-seq and snRNA-seq transcriptome data generated in this study are available in the Gene Expression Omnibus (GEO) under accession numbers GSE264303 and GSE271257. The mouse vagal sensory neuron scRNA-seq data are available in the GEO under accession number GSE124312. Source data are provided with this paper.

Code availability

The codes used for data analysis are available on the GitHub repository at https://github.com/Hweeseon/scRNA_VSGO/ under an MIT license.

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Acknowledgements

We thank S. Lee (Department of Biomedical Sciences, College of Medicine, Seoul National University) for technical support in the differentiation of VSGOs. We also thank S. Kim (Department of Biomedical Sciences, College of Medicine, Seoul National University) for technical support in the maintenance of iPS cells. We thank H. Cho and S. Yu (Genomic Medicine Institute, Medical Research Center, Seoul National University) for technical support in scRNA-seq. NNC0090-1170 (Liraglutide) was provided by Novo Nordisk Compound Sharing. We thank Geninus Inc. for performing sequencing process of scRNA-seq at i-VSN. We thank Macrogen Inc. for performing sequencing process of scRNA-seq of OEPD, EpP and snRNA-seq of VSGOs. This research was supported by a grant of the MD-PhD/Medical Scientist Training Program through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI21C1212) to K.A. This work was supported by Samsung Research Funding and Incubation Center of Samsung Electronics under Project Number SRF-IT1901-51 to S. Chung. This work was supported by the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (2022M3A9B6018217) to S. Chung. This work was supported by the NRF Grant funded by the Korean Government (MSIT) (No. 2018R1A5A2025964) to S.J.K. This research was supported by Basic Science Research Program through the NRF funded by the Ministry of Education (2020R1A6A1A03047972) to J.-I.K. Additionally, this research was supported by a grant of the Korea Dementia Research Project through the Korea Dementia Research Center (KDRC), funded by the Ministry of Health and Welfare and Ministry of Science and ICT, Republic of Korea (grant number: RS-2020-KH106747) to I.M.-J.

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Author notes

  1. These authors contributed equally: Kyusik Ahn, Hwee-Seon Park, Sieun Choi.

Authors and Affiliations

  1. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea

    Kyusik Ahn, Hwee-Seon Park, Hojeong Lee, Hyunjung Choi, Seok Beom Hong, Jihui Han, Jong Won Han, Jaehoon Song, Sang Jeong Kim, Jong-Il Kim & Inhee Mook-Jung

  2. Convergence Dementia Research Center, Seoul National University College of Medicine, Seoul, Republic of Korea

    Kyusik Ahn, Hyunjung Choi, Seok Beom Hong, Jihui Han, Jong Won Han, Jaehoon Song & Inhee Mook-Jung

  3. Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, Republic of Korea

    Hwee-Seon Park, Hyunjung Choi, Kyunghyuk Park, Bukyung Cha & Jong-Il Kim

  4. KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea

    Sieun Choi, Minseop Kim, Hyeonggyu Song & Seok Chung

  5. Department of Physiology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Hojeong Lee & Sang Jeong Kim

  6. School of Mechanical Engineering, Korea University, Seoul, Republic of Korea

    Jinchul Ahn, Hui-Wen Liu & Seok Chung

  7. Memory Network Medical Research Center, Neuroscience Research Institute, Wide River Institute of Immunology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Sang Jeong Kim

  8. Center for Brain Technology, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea

    Seok Chung

  9. Neuroscience Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea

    Inhee Mook-Jung

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  1. Kyusik Ahn
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Contributions

K.A. and I.M.-J. conceptualized the research. K.A. designed protocol for differentiating VSGOs. K.A. performed RT−qPCR, ICC of OEPD, EpP, VMN, HCO and bundle chip experiments. K.A. and J.S. performed ICC of VSGOs. I.M.-J. and H.C. supervised the overall experimental process. S.B.H. and H.C. modified previous protocol for differentiating HCO from iPS cells. H.L. and S.J.K. performed and analyzed patch-clamp analysis. J.H., S. Choi and K.A. performed PRV analysis. J.W.H. and K.A. performed calcium channel imaging of VSGO. K.P., B.C., H.-S.P. and J.-I.K. constructed scRNA libraries. H.-S.P. analyzed scRNA-seq data and wrote the method of analyzing scRNA-seq data. J.A., S. Choi, and S. Chung designed bundle chips. S. Choi, M.K., H.-W.L. and H.S. cultured VSGOs and HCO at bundle chips. S. Choi wrote methods for bundle chips. S. Choi and J.A. performed calcium channel imaging of bundle chip. K.A., H.-S.P., S. Choi and I.M.-J. wrote the final version of the paper.

Corresponding authors

Correspondence to Seok Chung, Jong-Il Kim or Inhee Mook-Jung.

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Competing interests

K.A. and I.M.-J. are inventors on a filed patent related to this study. The other authors declare no competing interests.

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Peer review information

Nature Methods thanks Keqiang Ye and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Graphical abstract of entire experimental process.

VSGO, Visceral sensory ganglion organoid; iPSC, induced-pluripotent stem cell; EB, Embryonic body; OEPD, Otic-epibranchial progenitor domain; EpP, Epibranchial placode; VSN, visceral sensory neuron; RAP, Receptor-associated protein; HCO, human colon organoid; Aβ, amyloid beta; VMN, visceral motor neuron; NC, negative control; CN, cognitively normal; sAD, sporadic Alzheimer’s disease.

Extended Data Fig. 2 Comparing diverse methods for differentiating EpP from OEPD.

(a) Schematic of diverse protocols tested for differentiating EpP from OEPD. (b-d) RT-PCR results of PHOX2B (b), PAX2 (c), and PAX8 (d) for comparing diverse protocols (n = 4 for each formula, all n: biological replicates). two-sided ANOVA with Tukey’s post-hoc test *P < 0.05, **P < 0.01, ***P < 0.001. All data were presented as mean ± SEM.

Source data

Extended Data Fig. 3 Functional characterization of VSGOs.

(a) Representative live-cell images before and after treatment with capsaicin (10 µM) and liraglutide (10 µM) (Baseline indicates before treatment) (Representative images among 2 batches, Scale bars: 100μm). (b) Schematic of timeline for calcium imaging. (c) Venn diagram representing number of i-VSNs activated by capsaicin or liraglutide or both. (d) Schematic of timeline for patch-clamp recording. (e) Bright field image showing patch-clamp experiment on VSN (Representative image among 3 batches, Scale bars: 10μm). (f) The examples of voltage traces. (g) Increasing relationship of spike number vs current (total 26 cells analyzed among 3 batches, mean ± SEM). (h and i) i-VSN was activated responding to capsaicin (h) and GLP1R agonist DMB (i). (j) Venn diagram of i-VSN number ratio responding to capsaicin or DMB, or both.

Extended Data Fig. 4 scRNA-seq label transfer and trajectory analysis.

(a-f) UMAPs of representative markers for each cluster identified at VSGOs, VSN for SLC17A6, SC for MPZ, SGC for TRPM3, GP for FBLN5 and MKI67, EpPL for PAX2. (g) comparison of VSN proportions between VSGO and i-VSN, two-sided Mann-whitney’s U test (4 biological replicates /each group, mean ± SEM). (h) Expression matrixplot of genes among iVSN cultured days (x axis). (i) UMAP of mouse nodose ganglion and VSGOs. (j) List of gene membership among each Macrogenes. (k) Expression of VSN score 2 among each VSN subclusters. (l) Terminal states of trajectory analysis. (m) Directed partition-based graph abstraction (PAGA) cluster UMAP. (n, o) UMAP of pseudotime and latent time. (p) UMAP plots of random walks among each cluster. (q, r) Smoothed gene expression trends for VSN_1 (q) and VSN_2 (r). Along with each trends, driver genes for each cell states are presented.

Source data

Extended Data Fig. 5 3D Axis-on-a-chip for identifying cell-cell interactions.

(a) Entire immunostaining image of axis-on-a-chip indicating VSGO chamber by TUJ1 (red) and HCO chamber by DSG2 (yellow) (Representative image among 3 biological replicates). (b and c) Diffusion test was conducted in microfluidic chip using 10 µM of 40 kDa FITC-dextran solution. Fluorescence images showing diffusion of dextran within the microchannel when with no ECM hydrogel or with ECM hydrogel composed of Collagen I and matrigel. (d) Graph represents relative intensity comparison of dextran fluorescence of each condition. (e) PRV-infected colon organoid connected with neurite bundle of VSGO. PRV expressing GFP and neurite immunostained with TUJ1 (red) (Representative image among 3 biological replicates). ECM, Extracellular matrix; PRV, pseudorabies virus. Scale bars indicate 500 µm (a), 50 µm (b), 200 µm (c and d).

Source data

Extended Data Fig. 6 VSN subcluster scRNA-seq classification and Axis-on-a-chip scRNA-seq.

(a) A comprehensive comparison of various characteristics of VSN clusters revealed between our data and previously published results. (b) UMAPs for canonical markers of HCO clusters or previously described (c) Ligand-receptor interaction plot between HCO and VSN. (d) Expression of VSN score 2 among previous VSGO clusters and chip VSN 3 and 5.

Extended Data Fig. 7 ICC images of axis-on-a-chip used for Aβ and tau propagation assay.

VSGO and its neurite bundle immunostained with TUJ1 (red) and HCO with DSG2 (yellow). (a) Images of propagated FITC-oAβ (a) (Representative images among E3 n = 7, E4 n = 4, AD4 n = 3) and Atto-488 tauPFF (b) (Representative images among E3 n = 5, E4 n = 4, AD4 n = 3) via the bundle of each E3, E4, and AD4 line. All n are biological replicates. Scale bars indicate 500μm.

Extended Data Fig. 8 Investigating ApoE4 and LRP1 in Aβ and Tau Propagation through Transcriptomic Comparisons and RAP Inhibition.

(a) Quantifications of proportions of identified VSN subclusters among E3 and E4 VSN. (b) UMAP presenting E3 and E4 sample for VSN cluster. (c, d) UMAPs for APOE (c) and LRP1 expression (d) among VSN cluster. (e-g) Expressions of gene sets among GO term: Cholesterol Biosynthetic process (e), Regulation of Autophagy (f) and Chromatin Organization (g) among each sample. (h) Schematic explaining mechanism of RAP working on LRP1 of i-VSN neurite. Representative images of FITC-oAβ (i) and Atto-499 tauPFF (k) before and after treatment of RAP on i-VSN (TUJ1, yellow). Normalized propagation ratio of FITC-oAβ (j) (n = 9 for E4 RAP- and n = 11 for E4 RAP+ among 4 independent differentiations each) and Atto-488 tauPFF (l) (n = 10 for E4 RAP- and n = 12 for E4 RAP+ among 4 independent differentiations each) were shown as bar graphs. RAP, receptor-associated protein. Scale bars: 20 µm. (a) two-sided chi-square test (j and k) two-sided student t test. *P < 0.05, ***P < 0.001. All data were presented as mean ± SEM.

Source data

Extended Data Fig. 9 Comparative evaluation of Aβ and tau propagation in VSN versus VMN.

(a) Timeline of protocol used for generating VMN from iPSCs. (b) ICC characterization of VMN expressing TH (green) and CHAT (red). (c-f) Aβ or tau propagation in VSN neurite bundle compared to VMN. Rendering images of FITC-oligomeric Aβ (c) and Atto-488 tauPFF (e). Bar graphs showing normalized propagation ratio of FITC-oligomeric Aβ (d) and Atto-488 tauPFF (f). VMN, visceral motor neuron. n = 4 independent differentiations among three batches. Scale bars indicate 50 µm (c and e). (d, f) Two-sided Mann-Whitney’s U test. *P < 0.05. All data were presented as mean ± SEM.

Source data

Extended Data Fig. 10 Correlation analysis between axis-on-a-chip propagation analysis and clinical data of donors.

(a-e) Correlation analysis between donor’s clinical data and quantified amyloid or tau propagation amount at axis-on-a-chip. Donor data: a, MMSE-Z score; b, Semantic memory Z-score; c, Global amyloid PET SUVR; d, Brain MRI hippocampal adjusted volume (HVA); e, FDG PET SUVR at PCC (n=16). Each n are biological replicates. Used iPSC lines: 1-control, 4-sAD lines. (a-e) Kendall’s tau correlation analysis. ***P<0.001.

Supplementary information

Supplementary Information

Supplementary Figs. 1–13, Tables 1–9 and Protocol.

Reporting Summary

Supplementary Table 10

Lists of gene membership among each Macrogenes.

Supplementary Table 11

Additional data for Trajectory analysis.

Supplementary Video 1

Live-cell calcium imaging of VSGOs after treating with liraglutide.

Supplementary Video 2

Live-cell calcium imaging of VSGOs after treating with capsaicin.

Source data

Source Data Fig. 1

Image quantification, RT–PCR source data.

Source Data Fig. 2

Image quantification source data.

Source Data Fig. 3

scRNA-seq cell count data.

Source Data Fig. 4

Live-cell calcium imaging source data.

Source Data Fig. 5

Image quantification source data.

Source Data Fig. 6

Image quantification source data.

Source Data Extended Data Fig. 2

RT–PCR source data.

Source Data Extended Data Fig. 4

scRNA-seq cell count source data.

Source Data Extended Data Fig. 5

Diffusion test source data.

Source Data Extended Data Fig. 8

Image quantification source data.

Source Data Extended Data Fig. 9

Image quantification source data.

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Ahn, K., Park, HS., Choi, S. et al. Differentiating visceral sensory ganglion organoids from induced pluripotent stem cells. Nat Methods 21, 2135–2146 (2024). https://doi.org/10.1038/s41592-024-02455-8

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