Abstract
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.
Highlights
Efficient generation of human induced pluripotent stem cell-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
We and others have previously shown that dual-smad inhibition of the BMP/TGFβ signaling pathways in definitive endoderm derived from induced pluripotent stem cells (iPSCs) and ESCs promotes the development of endoderm competent to form anterior foregut derivatives, such as NKX2-1 positive lung or thyroid lineages[10,11,12,13,23,24]
Prior single-cell sequencing of day 15 progenitors revealed the presence of cells expressing non-lung endodermal markers, including CDX2, and these non-lung lineages were enriched in the NKX2-1 negative fraction of cells
Summary
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. The efficient generation of iPSC-derived human intestinal organoids (HIOs) serves as a relevant tool to study development, but has great potential for patient-specific in vitro disease modeling and high-throughput drug screening applications. Distal patterning of iPSCderived HIOs to generate SATB2+ colonic organoids was achieved through BMP2 stimulation[20] These factors have all been shown to play a role in intestinal specification and epithelial proliferation during embryonic development[21]. We report the generation of a hiPSC CDX2-GFP reporter line that highlights the role of CDX2 as a specific marker for the emergence of iPSCderived intestinal progenitors This platform enables the study of both normal development as well as disease states of the gut (exemplified by cystic fibrosis), supporting the generation of patient-specific iPSC-derived organoids for interrogation, genetic manipulation, and large-scale drug screening applications
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