Establishment of Microvillus Inclusion Disease (MVID) human models using small intestinal enteroids and embryonic stem cells

Abstract

Microvillus Inclusion Disease (MVID) is a severe genetic disease resulting from loss‐of‐function mutations in the apical recycling associated actin motor, myosin Vb (Myo5B). MVID affects newborns (early‐stage) or infants (late‐stage), resulting in uncontrolled secretory diarrhea, and death. The diagnostic and pathognomonic features are found in enterocytes of the small intestine, specifically in the duodenum. Our laboratory investigates how apical ion transporter trafficking and signaling in the intestine results in MVID diarrhea. Treatment is unavailable due to limited understanding of MVID pathogenesis, and, access to human MVID intestinal tissues for research is not feasible due to the severity of illness in affected children. Therefore, generating appropriate research models is critical to understanding the disease and development of therapies.Our laboratory generated cultured intestinal cell models lacking Myo5B (lentivirus shRNA knockdown) for MVID studies (Kravtsov DV et al. 2016). Although cancer‐derived cell lines remain valuable, superior cutting‐edge technologies and models, such as enteroids/organoids and CRISPR/Cas 9 gene editing, are now available. Since MVID is an epithelial disease resulting in ion transporter defects in the proximal small intestine, we hypothesized that successful development of a human small intestinal model of MVID will provide a superior model for studies of MVID pathogenesis and treatment. Our goal is to develop complementary human intestinal models of MVID by impeding Myo5B function in both human duodenal enteroids (hDEs) and human embryonic stem cells (hESCs) that will be differentiated into small intestinal organoids.First, Myo5B will be silenced in 2D hDEs using lentiviral transfection as before. Immunoblot analysis will confirm Myo5B knockdown and enteroids will be characterized for expression, location, and function of ion transporters and proteins involved in MVID pathogenesis. A complementary MVID model will employ H9 hESCs for gene editing of the Myo5B single nucleotide polymorphism, G1125A, using CRISPR/Cas 9 technology. Clones will be sequenced to confirm successful gene editing of Myo5B. Once confirmed, H9 hESCs will be differentiated into small intestine and cultured into 3D intestinal organoids and characterized for MVID.To date, we have cultured undifferentiated (immature) and differentiated (mature) hDEs replicating crypt and villus enterocytes of the duodenum. For H9 hESC studies, the donor DNA guide, forward and reverse single guide RNAs were designed (http://crispr.mit.edu). Guide RNAs and donor DNA were transfected into H9 hESCs and clones sequenced to confirm Myo5B G1125A gene editing. H9 Myo5B G1125A cells will be differentiated into small intestine using an established protocol by McCracken KW et al. 2013. Successful establishment of these models will facilitate studies of ion transporter trafficking and signaling, and screening of compounds for treatment of MVID using physiologically relevant human intestinal models.Support or Funding InformationFunding: R01 DK077065This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.