A Novel Planar Crypt Microarray for Real-Time Evaluation of Human Intestinal Stem Cell Fate

PROJECT SUMMARY
Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis, affect an estimated 3.1
million U.S. adults. IBD pathophysiology is complex and involves genetic, environmental, and immune responses
as well as alterations in gut microbial short-chain fatty acid (SCFA) levels. Recent studies from the Magness lab
in murine ileal organoids demonstrated that exposure to IL-22 favors progenitor cell expansion over intestinal
stem cell (ISC) self-renewal, and this phenotype is thought be regulated by activating Signal Transducer and
Activator of Transcription (STAT)-3. Further, new evidence has shown that the gut microbial SCFA, butyrate,
enhances IL-22 receptor expression and IL-22-induced activation of STAT3 in human colon cancer cells. While
mouse, organoid, and cancer cell lines have helped to advance the field, more physiologically relevant in vitro
human models are needed to increase accuracy and resolution of experiments aimed at predicting and
uncovering the response of human gut ISCs and differentiated epithelium to such IBD-relevant factors.
My aims will use a novel planar crypt-microarray (PCM) device and reporter cell lines to live-image primary
human colonic stem cells during their response to IBD-related perturbations. PCMs have already been optimized
for culture of mouse ISCs, and preliminary data demonstrates human ISC compatibility. Aim 1 will adjust factors
such as PCM membrane composition, thickness, and timing of apical/basal media changes to promote confluent
monolayers and compartmentalization of stem (Olfm4-EGFP) and differentiated (Muc2-BFP) cell zones. Using
human-optimized PCMs, I will test the central hypothesis that butyrate-stimulated IL-22 signaling enhances
asymmetric division and differentiation in human colonic ISCs. Aim 2 will use CRISPR/Cas9 indels to
induce a frameshift loss-of-function (LOF) mutation in IL-22 receptor subunit alpha 1 into a tetracycline-inducible
H2B::RFP (tetO-H2B::RFP) human colon stem cell line to test whether IL-22 signaling is necessary for
asymmetric division. The tetO-H2B::RFP fusion protein allows for controlled, sensitive analysis of cell division
dynamics in mammalian cells, with rapidly dividing cells corresponding to diluted RFP fluorescence intensity over
time. RFP label-retaining cells will be assessed for phosphorylated STAT3 (pSTAT3) via immunofluorescence
imaging on unsorted cell monolayers and for perturbed lineage pathways by FACS-scRNA-seq. The LOF model
will be compared against IL22RA1+ controls on PCMs to determine if asymmetric division is enhanced by
butyrate-stimulated IL-22 signaling. Our lab already has preliminary data for Olfm4-EGFP and H2B reporter lines.
In the outstanding training environment in Dr. Scott Magness’ group at the University of North Carolina at Chapel
Hill, I will gain expertise in microfabrication and intestinal stem cell biology and learn new techniques in primary
human colon stem cell culture, single-cell biology/genomics, and bioinformatics. My career development training
plan focuses on research skills, professional development, and clinical skills to promote my path to independence
as an academic physician-scientist.