A HUMAN IPSC-DERIVED SCREENING MODEL FOR CROHN’S DISEASE DEMONSTRATES THE POTENTIAL ROLE OF JAK INHIBITORS IN MITIGATING INTESTINAL FIBROSIS

Abstract INTRODUCTION Intestinal fibrosis is a serious complication of Crohn’s disease (CD) and is caused by the excess deposition of extracellular matrix (ECM) by intestinal myofibroblasts. Currently, there are no approved treatments. Identification of potential treatments is impeded by intestinal myofibroblasts being difficult to obtain and having a limited lifespan in vitro. Human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs) contain a mesenchymal cell population that is analogous to intestinal myofibroblasts. iPSCs can be generated from peripheral blood mononuclear cells (PBMCs) and proliferate indefinitely meaning an unlimited number of patient-specific mesenchymal cells can be obtained. Our goal was to use our model of CD patients’ iPSC-derived mesenchymal (iPSC-MES) cells for high-throughput screening (HTS) and compare that their responses were similar to their paired intestinal fibroblasts of each patient. Methodology PBMCs and biopsy-derived (BD) ileal and colonic fibroblasts were obtained from 6 CD patients with a history of intestinal fibrosis. All PBMCs were reprogrammed to iPSCs, directed to HIOs and the mesenchymal cell population was purified. These cells were treated with TNFα/TGFβ (TT) for 48hrs to induce ECM deposition, and the antifibrotic potential of various therapeutics was examined. Selection of potential hits compared the z-score (z) of the compounds against baseline fibrosis (TTz=0), and a significant change was defined as a z ≥|±2|. Selected hits were then examined in BD ileal and colonic fibroblasts. RESULTS Our fibrosis model of iPSC-MES found a robust antifibrotic effect from the JAK1/2 inhibitor ruxolitinib (RXz=-4.91), in all 6 iPSC-MES lines. RX produced a significant reduction in ECM deposition in iPSC-MES cells (-34.81%) and similarly in patient matched BD-ileal (-53.76%) and -colonic fibroblasts (-43.14%) (Fig.1). Moreover, proteome enrichment analysis on iPSC-MES found that while TT produced a significant increase in a fibrosis-associated pathways, RX resulted in a decrease, namely: assembly of collagen fibrils (TTNES=4.80; RXNES=-2.83) and ECM organization (TTNES=5.39; RXNES=-2.65). We then evaluated other JAK inhibitors currently prescribed for the treatment of IBD: filgotinib (FG), tofacitinib (TF) and upadacitinib (UPA). While FG (-21.13%) and TF (-16.30%) showed a significant reduction in ECM, UPA (-39.22%) had the strongest antifibrotic activity, similar to RX (Fig.2). CONCLUSION We demonstrate the feasibility of utilizing iPSC-MES cells for HTS to identify potential antifibrotic therapies. This approach overcomes the limitations of biopsy-derived myofibroblasts, illustrates the potential utility of JAK inhibitors and thus opens up a new more feasible approach for studying precision-based treatments.