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Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID.
Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions.
Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin–positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane.
Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.
Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.