Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells. The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-α, TGF-ß, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

Generating and Utilizing Murine Cas9-Expressing Intestinal Organoids for Large-Scale Knockout Genetic Screening.

Organoid culture faithfully reproduces the in vivo characteristics of the intestinal/colon epithelium and elucidates molecular mechanisms underlying the regulation of stem cell compartment that, if altered, may lead tumorigenesis. CRISPR-Cas9 based editing technology has provided promising opportunities for targeted loss-of-function mutations at chosen sites in the genome of eukaryotes. Herein, we demonstrate a CRISPR/Cas9-mediated mutagenesis-based screening method using murine intestinal organoids by investigating the phenotypical morphology of Cas9-expressing murine intestinal organoids. Murine intestinal crypts can be isolated and seeded into Matrigel and grown into stable organoid lines. Organoids subsequently transduced and selected to generate Cas9 expressing organoids. These organoids can be further transduced with the second lentiviruses expressing guide RNA (gRNA) (s) and screened for 8-10 days using bright-field and fluorescent microscopy to determine possible morphological or phenotypical abnormalities. Via phenotypical screening analysis, the candidate knockouts can be selected based on differential abnormal growth pattern vs their untransduced or lenti-GFP transduced controls. Further assessment of these knockout organoids can be done via phalloidin and propidium iodide (PI) staining, proliferation assay and qRT-PCR and also biochemical analysis. This CRISPR/Cas9 organoid mutagenesis-based screening method provides a reliable and rapid approach for investigating large numbers of genes with unknown/poorly identified biological functions. Knockout intestinal organoids can be associated with the key biological function of the gene(s) in development, homeostasis, disease progression, tumorigenesis, and drug screening, thereby reducing and potentially replacing animal models.

Repurposing Antibacterial AM404 as a Potential Anticancer Drug for Targeting Colorectal Cancer Stem-Like Cells.

Tumour-promoting inflammation is involved in colorectal cancer (CRC) development and therapeutic resistance. However, the antibiotics and antibacterial drugs and signalling that regulate the potency of anticancer treatment upon forced differentiation of cancer stem-like cell (CSC) are not fully defined yet. We screened an NIH-clinical collection of the small-molecule compound library of antibacterial/anti-inflammatory agents that identified potential candidate drugs targeting CRC-SC for differentiation. Selected compounds were validated in both in vitro organoids and ex vivo colon explant models for their differentiation induction, impediment on neoplastic cell growth, and to elucidate the mechanism of their anticancer activity. We initially focused on AM404, an anandamide uptake inhibitor. AM404 is a metabolite of acetaminophen with antibacterial activity, which showed high potential in preventing CRC-SC features, such as stemness/de-differentiation, migration and drug-resistance. Furthermore, AM404 suppressed the expression of FBXL5 E3-ligase, where AM404 sensitivity was mimicked by FBXL5-knockout. This study uncovers a new molecular mechanism for AM404-altering FBXL5 oncogene which mediates chemo-resistance and CRC invasion, thereby proposes to repurpose antibacterial AM404 as an anticancer agent.

Morphological alterations of cultured human colorectal matched tumour and healthy organoids.

Organoids have extensive applications in many fields ranging from modelling human development and disease, personalised medicine, drug screening, etc. Moreover, in the last few years, several studies have evaluated the capacity of organoids as transplantation sources for therapeutic approaches and regenerative medicine. Nevertheless, depending on the origin of the cells and anatomical complications, an organoid transplant may make tissue regeneration difficult. However, some essential aspects of organoids including the morphological alterations and the growth pattern of the matched tumour and their healthy derived organoids have received less attention. Therefore, the current work focused on culturing matched healthy and tumour organoids from the same patient with colorectal cancer (CRC) and assessed their timed growth and structural differences on a daily basis. The healthy organoids underwent proliferation and branching morphogenesis, while the tumour organoids did not follow the same pattern, and the majority of them developed cystic structures instead. However, the number and size of tumour organoids were different from one patient to another. The differential morphological changes of the healthy versus human colonic tumour organoids likely linked to distinct molecular and cellular events during each day. Thus, while their specific structural features provide valuable in vitro models to study various aspects of human intestinal/colon tissue homeostasis and CRC which avoid or replace the use of animals in research, this model may also hold a great promise for the transplantation and regenerative medicine applications.