Crohn's disease and ulcerative colitis (UC) are multifactorial diseases which may involve several factors including: genetic predisposition, epithelial barrier integrity, innate and adaptive immune cell driven inflammation as well as changes in the patient's microbiome. The majority of in vitro models used to study the pathogenesis of these diseases do not address this complexity. Our lab has previously published a protocol for generation of intestinal tissue in vitro (Human Intestinal Organoids or HIOs) from human pluripotent stem cells (Spence et al. 2011, Mc Cracken et al. 2012). This protocol is limited because these HIOs lack the regional identity of intestinal tissue affected by Crohn's and Colitis, the ileum and colon. Furthermore, these HIOs lack innate and adaptive immune cells which can perpetuate inflammation in IBD. Here we present a method for generating both HIOs and Human Colonic Organoids (HCOs) which contain innate immune cells capable of mounting an inflammatory response. HCOs have both monocyte and granulocyte lineages present within the stromal compartment of the organoid. The generation of HCOs with macrophages and neutrophils will allow interrogation of pathways regulating inflammation as well as the interactions between epithelial cells and innate immune cells.Methods:
HIOs and HCOs were generated by modifying our previously published protocol. RNA-seq data was used to identify an immune cell signature in HCOs. Expression of PU.1 and CD68 organoids was confirmed by immunofluorescence staining and confocal imaging using human biopsy samples as controls. Cytokine expression was measured using Luminex arrays. Lipopolysaccharide (LPS) induced necrosis was analyzed using wholemount confocal microscopy.Results:
We have engineered HCOs containing immune cells expressing PU.1, a marker of both primitive and definitive hematopoietic cells. In addition, HCOs contain cells which express the macrophage markers CD14, CD68 and C163 as detected by RNA-seq. A 3 days treatment of engineered HCOs with media containing inhibitors of p38 MAPK and TGF-B signaling, as well as leu-Gastrin and Nicotinamide (SAGN), also allows the generation of cells which express myeloperoxidase (a neutrophil marker) suggesting engineered HCOs contain precursors of both the monocyte and granulocyte lineage. Interestingly HCOs express significantly higher levels of the inflammatory cytokines IL-6, IL-8, CCL3 and CCL4 (as measured by Luminex Arrays) compared to HIOs but did not express higher levels of TNFa. In addition, engineered HCOs are hyper-responsive to stimulation with LPS and become increasingly necrotic when compared to HIOs. This is consistent with the increased expression of the LPS co-receptor CD14 in HCOs compared to HIOs.Conclusions:
The generation of HCOs from human pluripotent stem cells will allow the study of complex cell-cell interactions which may be relevant to pathogenesis of IBD. Since HCOs can be generated from Induced Pluripotent Stem Cells (IPSCs), HCOs can potentially be generated from colitis patients with specific mutations to determine the effect of the mutated gene on innate immunity, mesenchymal signaling and epithelial integrity. In addition, since HCOs display inflammation without external triggers, they could be used for drug screening.