Abstract
Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. Although the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe a protocol to differentiate hPSCs into different types of organ-specific mesoderm, leveraging signaling networks and molecular markers elucidated from single-cell transcriptomics of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm treated with either retinoic acid (RA) or RA together with a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm, respectively, after 4-d cultures. These are directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4 to 7 in cultures. By day 7, the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60–90% pure liver septum transversum/mesothelium-like, 70–80% pure liver-like fibroblasts and populations of ~35% respiratory-like mesoderm, gastric-like mesoderm or esophageal-like mesoderm. This protocol can be performed by anyone with moderate experience differentiating hPSCs, provides a novel platform to study human mesoderm development and can be used to engineer more complex foregut tissue for disease modeling and regenerative medicine.
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Data availability
The main data discussed in this protocol were generated as part of the studies published in the supporting primary research papers14. The raw datasets of qRT-PCR and immunostaining have been deposited in the online open access figshare repository (https://doi.org/10.6084/m9.figshare.19723189). The published mouse foregut scRNA-seq data used to infer the signaling roadmap is available from the Gene Expression Omnibus database under accession code GSE136689. The scRNA-seq data can also be explored at https://research.cchmc.org/ZornLab-singlecell/.
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Acknowledgements
We thank P. Chaturvedi for generating plots from the mouse scRNA-seq data. We thank all members in the Zorn, Takebe, Wells and Morimoto laboratories for reagents and feedback. We also thank C. Mayhew and A. Pitstick from the Pluripotent Stem Cell Facility as well as M. Kofron and E. Meyer from the Confocal Imaging Core at Cincinnati Children’s Hospital for constant support and guidance. This work was supported by grant NICHD P01HD093363 to A.M.Z. and J.M.W., the NIH Director’s New Innovator Award (DP2 DK128799-01) (to T.T.), the New York Stem Cell Foundation (to T.T.) and by a CCHMC CURE award to A.M.Z., J.M.W. and T.T. K.K. is supported by a Uehara Memorial Foundation postdoctoral fellowship and a fund for the Promotion of Joint International Research (A) (18KK0423). K.I. is supported by the Japan Society for the Promotion of Science Overseas Research Fellowship.
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K.K., L.H., J.M.W., T.T. and A.M.Z. conceived the project. K.K. and A.M.Z. wrote the manuscript with support from K.I., M.M., J.M.W. and T.T. K.K., K.I., A.S. and C.F.-H. performed experiments, and K.K. and K.I. analyzed data. A.M.Z. supervised the project.
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K.K., L.H., M.M. and A.M.Z. have filed a patent application on this protocol. All other authors declare no competing interests.
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Nature Protocols thanks Jonaa Fowler, Kyle Loh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Han, L. et al. Nat. Commun. 11, 4158 (2020): https://doi.org/10.1038/s41467-020-17968-x
Eicher, A. K. et al. Cell Stem Cell 29, 36–51.e6 (2022): https://doi.org/10.1016/j.stem.2021.10.010
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Kishimoto, K., Iwasawa, K., Sorel, A. et al. Directed differentiation of human pluripotent stem cells into diverse organ-specific mesenchyme of the digestive and respiratory systems. Nat Protoc 17, 2699–2719 (2022). https://doi.org/10.1038/s41596-022-00733-3
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DOI: https://doi.org/10.1038/s41596-022-00733-3
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