Postdoctoral Scientist Cedars-Sinai Medical Center Los Angeles, California, United States
Abstract: Paneth cells (PCs) are specialized secretory epithelial cells crucial for intestinal homeostasis. They produce antimicrobial peptides (AMPs) to protect the intestinal epithelium from pathogens. Studying PCs can be challenging due to their inability to be maintained in long-term ex vivo culture systems and the limited protocols for their enrichment in human intestinal organoids (HIOs). To overcome this, we developed a strategy to enrich PCs in HIOs derived from induced pluripotent stem cells (iPSCs). iPSCs were directed to form HIOs, which were dissociated to generate epithelial-only HIOs (eHIOs). eHIOs were cultured in a proliferation medium containing EGF, Noggin, and CHIR99021, with γ-secretase inhibitor DAPT added to promote PC differentiation. PC enrichment was analyzed using single-cell RNA sequencing (scRNA-seq) to provide an overview of cell diversity and confirm PC enrichment. Quantitative PCR (qPCR), flow cytometry (FC), and immunocytochemistry (ICC) quantified PC numbers and AMP expression. Electron microscopy (EM) evaluated PC granule morphology, and LYZ ELISA assessed antimicrobial secretion. Our scRNA-seq data captured the expression of all well-characterized PC markers, providing a comprehensive transcriptomic profile that confirms the identity and enrichment of PCs in the system. FC showed an increase in the PC marker LYZ, from 1% in proliferation medium to 28% after DAPT treatment. DAPT treatment significantly upregulated PC-associated genes, including LYZ (log2 fold change: +0.83), DEFA5 and DEFA6 (log2 fold change: +18.93 and +7.72), and REG3A (log2 fold change: +11.07). ICC confirmed that DAPT-treated eHIOs were enriched in LYZ-positive granulated cells and expressed additional PC AMPs. EM showed electron-dense granules characteristic of PCs. Stimulation with muramyl dipeptide (MDP) induced lysozyme secretion (log2 fold change: +0.378), confirming functional activity. We developed an effective protocol for enriching PCs in iPSC-derived organoids. This system provides a robust model to study human PCs and enables the generation of patient-specific organoids with genetic variations. The enriched organoids retain functional activity, highlighting their potential for modeling intestinal disease mechanisms and evaluating patient-specific therapeutic strategies.
Funding Source: NIH/1R01DK123511, Mechanisms of TL1A-driven Paneth Cell dysfunction in IBD; California Institute for Regenerative Medicine (CIRM EDUC4-1275)
