A PERSONALIZED HUMAN PANETH CELL MODEL THAT IS RESPONSIVE TO MICROBES/MICROBIAL LIGANDS

Abstract INTRODUCTION Paneth cells are a specialized intestinal epithelial subtype whose major function is to produce antimicrobial peptides (AMPs). Alterations in Paneth cell function are associated with Crohn’s disease (CD) but the confounding influences of environmental factors and/or genetic variations makes it difficult to identify the cause of this. Human intestinal organoid (HIO) technology now allows for the generation of intestinal epithelium from any individual but there are relatively few protocols that allow for the enrichment of Paneth cells within organoids. Our goal was to specifically enrich the Paneth cell population in HIOs derived from induced pluripotent stem cells (iPSCs). METHODS iPSCs from a control individual were directed to HIOs and then dissociated to generate purified populations of epithelial only-HIOs (eHIOs). eHIOs were passaged weekly in proliferation medium (EGF, Noggin and CHIR99021) and the γ-secretase inhibitor, DAPT, was added to direct towards a Paneth cell fate. Flow cytometry, qPCR and immunocytochemistry were used to estimate Paneth cell number, gene expression and presence of various AMPs respectively. To confirm the functionality of this cell type, organoids were seeded onto Transwells to assess the responses to microbes/microbial ligands. RESULTS iPSC-derived eHIOs could be maintained for at least 4 months in proliferation medium. Flow cytometry analysis of the Paneth marker lysozyme revealed that the population of Paneth cells in eHIOs significantly increased from ∼1% in proliferation media to ∼30% upon treatment with DAPT. qPCR analysis demonstrated that DAPT treatment significantly increased the expression of the Paneth cell associated genes DEFA5, DEFA6, ITLN2, REG3A and PLA2G2A. Immunoctyochemistry revealed that DAPT treated eHIOs were enriched for granulated DEFA5, ITLN2 and REG3A in addition to lysozyme, and revealed the presence of the bacterial sensors TLR2, TLR5 and NOD2. Finally, this differentiation protocol was successfully established in transwell culture thereby permitting easy administration of peptides/live microbes to the apical aspect of this epithelial tissue. Addition of the NOD2 ligand, muramyl dipeptide (MDP), significantly induced lysozyme secretion and live bacteria, isolated from IBD patients, led to a significant increase in the secretion of DEFA5 as found via mass spectrometry. CONCLUSION We have successfully developed a methodology to enrich Paneth cells in iPSC-derived HIOs. Given that iPSCs can be generated from donor cells stored in well characterized biorepositories or from a small blood draw from any CD patient, this modeling system now opens a new avenue of research by allowing an examination of how environmental factors (microbes/cytokines) and/or genetic variations influence human Paneth cell function in a personalized manner.