Supplementary Figure 6: PEG-4MAL hydrogel serves as an injectable delivery vehicle in colonic mucosal wound model and promotes HIO engraftment.

(a) Mechanically-induced submucosal wounds were performed in the distal colon of mice using a mechanical probe through a mouse colonoscope. One day post-wounding HIOs generated in engineered 4% PEG-4MAL hydrogels or Matrigel^TM were recovered from the matrix, mixed with the engineered hydrogel precursor solutions, and injected underneath the submucosal wounds. A group with no injections, HIOs injected in saline, or injection of HIO-free hydrogel precursor solutions were used as control groups. Distal colon tissue harvest, immunostaining and imaging was performed 4 weeks post-wounding. (b) Fluorescence microscopy images labeled for human mitochondria (HUMIT) of murine colonic tissue at the wound site at 4 weeks post-injection or control tissue. DAPI, counterstain. “L” indicates HIO lumen. Bar, 100 μm. (c) In situ hybridization images of (c) control adult human colon or sections taken at the mouse colonic wound site stained for human OLFM4 + cells. (d) In situ hybridization images of tissue sections from mice colon that did not undergo colonic injuries or received HIO injections (control) and sections taken at the mouse colonic wound site stained for mouse Lgr5 + intestinal stem cells. Bar, 50 μm. Two independent experiments were performed and data is presented for one of the experiments. Experiments performed with 4 mice per experimental group (five colonic wounds/injections per mouse; b–d).