Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature

Charlie J Childs¹, Holly M Poling², Kevin Chen³, Yu-Hwai Tsai⁴, Angeline Wu⁴, Abigail Vallie⁵, Madeline K Eiken⁶, Sha Huang⁴, Caden W Sweet⁴, Ryan Schreiner⁷, Zhiwei Xiao⁴, Ryan C Spencer⁴, Samantha A Paris⁴, Ansley S Conchola⁵, Jonathan W Villanueva⁴, Meghan F Anderman⁴, Emily M Holloway⁸, Akaljot Singh⁹, Roman J Giger¹⁰, Maxime M Mahe¹¹, Claudia Loebel¹², Michael A Helmrath⁹, Katherine D Walton⁴, Shahin Rafii³, Jason R Spence¹³

Affiliations

¹ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI, USA.
² Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Biomedical Engineering, University of Cincinnati College of Engineering and Applied Science, Cincinnati, OH, USA.
³ Hartman Institute for Therapeutic Organ Regeneration, Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA.
⁴ Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
⁵ Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA.
⁶ Department of Biomedical Engineering, University of Michigan and University of Michigan College of Engineering, Ann Arbor, MI, USA.
⁷ Hartman Institute for Therapeutic Organ Regeneration, Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA; Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY, USA.
⁸ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA.
⁹ Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
¹⁰ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA.
¹¹ Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Université de Nantes, INSERM, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France.
¹² Department of Biomedical Engineering, University of Michigan and University of Michigan College of Engineering, Ann Arbor, MI, USA; Department of Materials Science and Engineering, University of Michigan College of Engineering, Ann Arbor, MI, USA.
¹³ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA; Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan and University of Michigan College of Engineering, Ann Arbor, MI, USA. Electronic address: spencejr@umich.edu.

PMID: 40043706
PMCID: PMC11973701 (available on 2026-04-03)
DOI: 10.1016/j.stem.2025.02.007

Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature

Charlie J Childs et al. Cell Stem Cell. 2025.

. 2025 Apr 3;32(4):640-651.e9.

doi: 10.1016/j.stem.2025.02.007. Epub 2025 Mar 4.

Authors

Affiliations

¹ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI, USA.
² Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Biomedical Engineering, University of Cincinnati College of Engineering and Applied Science, Cincinnati, OH, USA.
³ Hartman Institute for Therapeutic Organ Regeneration, Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA.
⁴ Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
⁵ Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA.
⁶ Department of Biomedical Engineering, University of Michigan and University of Michigan College of Engineering, Ann Arbor, MI, USA.
⁷ Hartman Institute for Therapeutic Organ Regeneration, Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA; Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY, USA.
⁸ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA.
⁹ Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
¹⁰ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA.
¹¹ Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Université de Nantes, INSERM, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France.
¹² Department of Biomedical Engineering, University of Michigan and University of Michigan College of Engineering, Ann Arbor, MI, USA; Department of Materials Science and Engineering, University of Michigan College of Engineering, Ann Arbor, MI, USA.
¹³ Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA; Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Biomedical Engineering, University of Michigan and University of Michigan College of Engineering, Ann Arbor, MI, USA. Electronic address: spencejr@umich.edu.

PMID: 40043706
PMCID: PMC11973701 (available on 2026-04-03)
DOI: 10.1016/j.stem.2025.02.007

Abstract

Human intestinal organoids (HIOs) derived from human pluripotent stem cells co-differentiate both epithelial and mesenchymal lineages in vitro but lack important cell types such as neurons, endothelial cells, and smooth muscle, which limits translational potential. Here, we demonstrate that the intestinal stem cell niche factor, EPIREGULIN (EREG), enhances HIO differentiation with epithelium, mesenchyme, enteric neuroglial populations, endothelial cells, and organized smooth muscle in a single differentiation, without the need for co-culture. When transplanted into a murine host, HIOs mature and demonstrate enteric nervous system function, undergoing peristaltic-like contractions indicative of a functional neuromuscular unit. HIOs also form functional vasculature, demonstrated in vitro using microfluidic devices and in vivo following transplantation, where HIO endothelial cells anastomose with host vasculature. These complex HIOs represent a transformative tool for translational research in the human gut and can be used to interrogate complex diseases as well as for testing therapeutic interventions with high fidelity to human pathophysiology.

Keywords: HIO; directed differentiation; enteric neruon; human intestinal organoids; intestine; organoids; pluripotent; stem cells; vasculature.

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Conflict of interest statement

Declaration of interests C.J.C., E.M.H., M.K.E., and J.R.S. hold intellectual property pertaining to human intestinal organoids. C.J.C., M.K.E., and J.R.S. are co-founders and own equity in Intero Biosystems, Inc.

References

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Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature

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Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature

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Abstract

Conflict of interest statement

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