O1121: Measuring Motility in Ipsc Derived Tissue-engineered Small Intestine

Background: Hirschprung disease (HD) results from failure of migration of enteric neural crest cells (ENCC) resulting in aganglionic distal intestine. HD is found in 1 in 5000 live births and one-third of patients with severe cases never achieve continence. Our lab has previously shown that Tissue-Engineered Small Intestine (TESI), which histologically and morphologically resembles native human intestine, can be reliably derived from human intestinal organoids (HIO) made from induced pluripotent stem cells (iPSC). This HIO-TESI develops without a nervous system, providing a model of human aganglionic intestine. When ENCC derived from iPSC are implanted into TESI, an enteric nervous system (ENS) develops and the resulting ENCC-HIO-TESI demonstrate restored contractile function. We hypothesized that motility could be measured with an automated video analysis system, and that these measurements might inform dose selection.

Methods: HIO derived from H9 iPSC were loaded onto biodegradable scaffolds and implanted into the omentum of immune compromised NOD/SCID mice. After ten weeks, ENCC derived from LiPSC-GR1.1 iPSC were implanted into the HIO-TESI at a concentration of either 1e6 or 1e7. Control specimens were injected with vehicle alone. After three months tissues were explanted and treated with methylene blue to inactivate confounding activity from interstitial cells of Cajal (ICC). Videos capturing the HIO-TESI contracting were recorded. Videos were calibrated to measure accurate distances and random point masses identified in Tracker, an open-source software that allows automated two-dimensional point mass capture. We further resolved x and y coordinates into single vectors, and calculated the amplitudes of contraction (Figure 1). Native mouse intestine served as an additional control. The explants were sectioned and stained for human and neuronal markers.

Results: ENCC-HIO-TESI demonstrated TUJ1- and LAMIN-positive cells in the intermuscular layer, indicating an ENS derived from human tissue, which resulted in contraction. The average maximum amplitude for the ENCC-HIO-TESI injected with 1e6 or 1e7 ENCC was approximately 1mm, which was greater than that of the controls at 0.2mm (p < 0.05) and mouse intestine. The amplitudes of the two experimental conditions were not statistically different from each other (Figure 2).

Conclusion: An automated calibrated video analysis of ENCC-HIO-TESI demonstrated periods of contraction and relaxation and quantified, for the first time, the amplitude of point mass motion for two different doses of ENCC in a model of human aganglionic intestine. ENCC-HIO-TESI demonstrated an ENS in the appropriate location on immunofluorescence, indicating that ENCC derived from iPSC can repopulate human aganglionic intestine. Quantifying the function of ENS derived from human iPSC that populate aganglionic intestine in an immunocompromised mouse model may inform the future dose for cell therapy and allow measurements of additional types of contractions, a motility index, and response to pro-motility pharmaceuticals.