Single cell RNA sequencing of day 6 and day 13 endodermal progenitors

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. Overall design: Here, we report a novel 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 novel CDX2eGFP iPSC “knock-in” reporter line to track the emergence of hindgut progenitors, we followed 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 employed 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.