Induced pluripotent stem cells as an innovative model for therapy personalization of inflammatory bowel disease.

Crohn’s disease (CD) is a chronic relapsing inflammatory bowel disease that may affect any part of the gastrointestinal tract but most commonly the ileum and the colon. The inflammation extends through the entire thickness of the bowel wall from the mucosa to the serosa. Thiopurines are drugs commonly used in Crohn’s disease (CD) even if some adverse effects are reported. In particular, we focused on the study thiopurine-induced pancreatitis (TIP), a severe and idiosyncratic adverse reaction that affects around 3-5% of CD patients treated with azathioprine, that leads to therapy interruption and could require ad hoc therapy with significative associated costs. Molecular mechanism of TIP is unknown and no validated biomarker is available to assist clinicians in preventing it. Induced pluripotent stem cells (iPSCs) are stem cells obtained reprogramming somatic cells using specific reprogramming factors. iPSCs maintain the donor genetic heritage and have become a powerful technique to model drug adverse effects in a personalized way. iPSCs can differentiate under adequate stimuli into almost every somatic lineage, representing an innovative model to study mechanisms of adverse drug reactions in individual patients' tissues not easily obtainable from human probands. At IRCCS Burlo Garofolo (Trieste, Italy) 3 pediatric CD patients that developed TIP and 3 after azathioprine treatment and 3 CD controls were enrolled and iPSCs were obtained reprogramming peripheral blood mononuclear cells in collaboration with Prof. Gliliani (Brescia, Italy). CD iPSCs were differentiated in pancreatic exocrine cells using the 4 stage protocol developed by Prof. Sasaki (Shinshu University, Japan). Each differentiation stage presents characteristic genetic expression markers: OCT4 is characteristic of undifferentiated cells (iPSCs), FOXA2 and SOX17 of definitive endoderm (stage I), PDX1 of pancreatic progenitors (stage III) and amylase, in particular its pancreatic isoforms AMY2A and AMY2B of pancreatic exocrine cells (stage IV). Differentiation efficiency was analyzed by PCR-real time and immunofluorescence techniques. The sensitivity to thiopurines of TIP and no-TIP CD patient-specific iPSCs and differentiated cells were investigated by MTT assay exposing cells to azathioprine, mercaptopurine and thioguanine for 72 hours. TIP patients iPSCs and pancreatic progenitors resulted more sensitive to mercaptopurine and thioguanine (mercaptopurine p= 0.0162, thioguanine p= 0.0012; two way ANOVA no-TIP vs TIP patients iPSCs; mercaptopurine p = 0.0174; thioguanine p = 0.0144; two way ANOVA no-TIP vs TIP patients pancreatic progenitors). All patients resulted wild type for TPMT polymorphisms letting us to conclude that the different sensitivity between no-TIP and TIP iPSCs and pancreatic progenitors was not related to TPMT genetic variants but to other mechanisms. Thiopurine effect is strictly correlated to cell proliferation being these drugs cell cycle-specific agents interfering during the S phase. iPSCs resulted extremely sensitive to thiopurines in comparison to differentiated cells and to a panel of immortalized lines including the H6C7 ductal pancreatic line. Analysis of cell cycle showed an higher percentage of cells in the S phase in CD-iPSCs with respect to the H6C7 line but not to definitive endoderm or pancreatic progenitors. The faster proliferation of CD-iPSCs well explains their higher sensitivity to thiopurines with respect to H6C7 however, the lower sensitivity of definitive endoderm and pancreatic progenitors cannot be explained basing on the different proliferation of these cells in comparison to iPSCs. The in vitro model established has proven to be suitable for studying and investigating TIP predisposition in a personalized way in pediatric CD patients and could be further developed to study other drugs causing pancreatitis in other diseases.