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Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip

Abstract

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial–mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.

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Fig. 1: Microwell chips for generating and culturing 3D cell aggregates.
Fig. 2: PDLO differentiation in the microwell chip.
Fig. 3: Apical-out polarity of the microwell chip-derived PDLOs switched upon orthotopic transplantation or embedding into Matrigel.
Fig. 4: scRNA-seq identifies cellular heterogeneity along the differentiation from pancreatic progenitors to PDLOs.
Fig. 5: Ductal subcluster-specific genes located within PLDOs and primary pancreas tissue on the protein level.
Fig. 6: Recovery of transcriptome dynamics predicts differentiation paths for duct-like cell types.
Fig. 7: Duct-like cells of the PDLOs clustered with primary ductal cells and resembled CFTR+-/mucin+-subpopulations.
Fig. 8: Potential PDAC biomarkers in the secretome and transcriptome of PDLOs.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Raw data, read counts and the analysed datasets from scRNA-seq can be accessed from the Gene Expression Omnibus repository using the accession code GSE162547. Mass spectrometry data have been deposited on the PRIDE database and can be accessed with the identifier PXD024461.

Code availability

The code for scRNA-seq analysis is available on Zenodo at https://doi.org/10.5281/zenodo.4738625.

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Acknowledgements

This work is supported by the Helmholtz Pioneer Campus, the German Ministry of Education and Research (INDIMED-PancChip), the Baden-Württemberg Stiftung (project ExPoChip), ERC (Consolidator Grant Number 772646), Deutsche Forschungsgemeinschaft (DFG) Sachbeihilfe KL 2544/7-1, KL 2544/1-2, KL 2544/5-1, GRK 2254/1 and 2, Heisenberg-Programm KL 2544/6-1, German Cancer Aid (Grant 111879), Else Kröner-Fresenius-Stiftung (supporting A.K. with an Excellence grant and M.H. with a First Application grant) and Bausteinprogramm of Ulm University (granted to M.H.). We thank NK-Optik for instrumental support. We thank M. Löhr (Karolinska Institute) for providing the human pancreatic stellate cells and T. Walzthöni for bioinformatics support provided at the Bioinformatics Core Facility, Institute of Computational Biology, Helmholtz Zentrum München.

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Contributions

S.W., M.B., M.H., A.K. and M. Meier designed the study. S.W. designed and produced the microwell chips, based on work from M. Moussus. S.W., M.B., J.M. and M.H. executed the biological experiments. S.W., M.B., M.H. and T.G. did the imaging and image analysis. S.W., M.S. and H.L. performed the scRNA-seq processing and S.W. did the analysis. C.v.T. and S.M.H. did the mass spectrometric measurements and data processing. S.E.W. and P.M. stained and evaluated the FLNB patient cohort. L.S. and T.S. took the serum samples of the PDAC patient cohort and M.H. performed the ELISA. S.W., M.B. and M. Meier analysed the MS/MS and FLNB screening results. M.H., A.K. and M. Meier received the funding and supervised the study. The manuscript was written by S.W., M.B., M.H., A.K. and M. Meier. All authors corrected and approved the paper.

Corresponding authors

Correspondence to Meike Hohwieler, Alexander Kleger or Matthias Meier.

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Peer review information Nature Biomedical Engineering thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Supplementary information

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Supplementary figures.

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Supplementary Dataset 1

Top 300 DEGs for the kinetic clusters in Fig. 4c.

Supplementary Dataset 2

Dynamical genes for Fig. 6f.

Supplementary Dataset 3

Top 300 DEGs for the Louvain clusters in Fig. 7a.

Supplementary Dataset 4

PDLO secretome and proteome.

Supplementary Dataset 5

Results of the ELISA FLNB screening corresponding to Fig. 8h.

Supplementary Dataset 6

Antibodies used for IHC/IF-p, ICC/IF and FC staining.

Supplementary Video 1

Live-cell imaging during PDLO differentiation from day 24 until day 31.

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Wiedenmann, S., Breunig, M., Merkle, J. et al. Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip. Nat Biomed Eng 5, 897–913 (2021). https://doi.org/10.1038/s41551-021-00757-2

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