Olivia Bay

germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

Abstract We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

Abstract INTRODUCTION Intestinal fibrosis is a serious complication of Crohn's disease (CD) and is caused by the excess deposition of extracellular matrix protein. There are no therapies to prevent or treat this and surgical intervention remains the only treatment option. Numerous cell types, including intestinal epithelial and mesenchymal cells, are implicated in this but research efforts are impaired by a lack of in vitro models. Human intestinal organoids (HIOs), derived from induced pluripotent stem cells (iPSCs), are comprised of both of these cell types; therefore iPSC-derived HIOs represent an approach in which an unlimited number of patient specific cells could be generated for such models. Our goal was to confirm the feasibility of using iPSC-derived epithelial and mesenchymal cells from CD patients to model intestinal fibrosis METHODS iPSCs from CD patients with and without fibrotic complications were directed to form HIOs. As iPSC-derived HIOs contain epithelial and mesenchymal cells, the organoids were dissociated to generate purified cultures of epithelial-only HIOs (eHIOs) and mesenchymal cells. Both cell types were cultured with TNFα and TGFβ, and responses were assayed after 8, 24 and 48 hrs. Further assessment on the transcriptomic profile of the cultures was conducted using the Nanostring nCounter Fibrosis Panel. RESULTS Mesenchymal cells treated with TNFα for 48 hrs showed a significant increase in MMP9 expression, whereas TGFβ elicited an upregulation in COL1A1 and COL5A1. Further analysis via Nanostring identified the upregulation of numerous other fibrosis-related genes in response to TNFα stimulation, mainly: CCL2, IL6, MMP7, and MMP9. In eHIO cultures, after 24 hrs of cytokine exposure, we detected a significant upregulation in EMT (NCAD, VIM, and SNAI2) and fibrosis genes (COL1A1, COL5A1, and MMP9), mainly in response to TGFβ and the combination TNFα/TGFβ. Nanostring further identified a total of 121 differentially expressed genes, mostly in response to these treatments, particularly: CCL2, IL11, MMP9, MMP10. Overall, Nanostring analysis found an upregulation in pathways associated with ECM production and remodeling, immunomodulation, and ligands associated with EMT and cell migration. CONCLUSION We have successfully generated a system to assay intestinal fibrosis in a patient-specific manner using iPSC-derived epithelial and mesenchymal cells from CD patients. Our model elicited a reliable and reproducible response to inflammatory and fibrogenic stimuli, measured by the differential expression of key fibrosis-associated genes. Future studies will focus on a broader analysis of these data via RNA-seq to investigate a potential fibrogenic phenotype and to identify novel genes/pathways that may play in role in the pathophysiology of intestinal fibrosis in CD.

Abstract INTRODUCTION Paneth cells are a specialized intestinal epithelial cell 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 and 2 CD patients 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 determine Paneth cell numbers, and gene and protein expression of various AMPs respectively. RESULTS iPSC-derived eHIOs from the control individual, which were used to develop this enrichment protocol, 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 ~28% 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. Immunocytochemistry revealed that DAPT treated eHIOs were enriched for cells possessing granulated lysozyme, and also for additional AMPs such as DEFA5, ITLN2 and REG3A. The presence of the bacterial sensors TLR2, TLR5 and NOD2 were also detected in our Paneth cells. Finally, to confirm the applicability to CD patients, we applied our protocol and found the population of Paneth cells in CD eHIOs significantly increased from ~1% in proliferation media to ~10% upon treatment with DAPT, had significantly increased expression of the Paneth cell related genes and were also were enriched for cells with granulated AMPs. CONCLUSION We have successfully developed a methodology to enrich Paneth cells in iPSC-derived HIOs from both healthy and CD patients. Given that iPSCs can be generated from donor cells stored in well characterized biorepositories or obtained from a small blood draw from any CD patient, this modeling system now opens up 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. Fluorescent image showing the presence of granulated DEFA5 (green) in E-cadherin+ (red) human iPSC-derived intestinal organoids treated with DAPT (X60). qPCR analysis of Paneth cell related genes in proliferation media compared to DAPT treatment. *P < .05, **P < .01, ***P < .001, and ****P < .0001 as compared to gene expression in proliferation media.