Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells

Aditya Mithal, Amalia Capilla,Dar Heinze, Andrew Berical, Carlos Villacorta-Martin, Marall Vedaie, Anjali Jacob, Kristine Abo, Aleksander Szymaniak, Megan Peasley, Alexander Stuffer, John Mahoney, Darrell N. Kotton, Finn Hawkins, and Gustavo Mostoslavsky
Source: Nat Commun.
Publication Date: (2020)
Issue: 11: 215
Research Area:
Cancer Research/Cell Biology
Stem Cells
Regenerative medicine
Cells used in publication:
H9 (derivative of HuT78)
Species: human
Tissue Origin: blood
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit

Gene editing of bBU1c2. Parental bBU1c2 iPSCs from one confluent six-well plate and 2 10 cm dishes were dissociated from their tissue culture vessels using ReLeSR (StemCell Technologies), and dissociated into single-cell suspensions. In total, 6 × 106 cells were nucleofected with 5 µg of guide plasmid DNA and 5 µg of donor plasmid DNA, and re-plated on fresh 10 cm Matrigel-coated dishes. The cells were nucleofected on an Amaxa™ 4D-Nucleofector™ using the Lonza Nucleofector P3 Primary Cell 4D-Nucleofector™ X Kit (V4XP-3024), as per the manufacturer’s instructions, on the “HESCell H9” program.


Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications.