S. M. Zuber1, G. Levin1, C. R. Schlieve1, A. I. Squillaro1, K. L. Fowler1, L. A. Nucho1, T. C. Grikscheit1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction:
Intestine organoid units (OU), multicellular epithelial and mesenchymal cell clusters, include stem and progenitor cells and can be cultured long-term in a reduced-factor medium with subsequent generation of tissue-engineered small intestine (TESI) after implantation in vivo. Application of Y-27632 (Rho-Kinase Inhibitor), A-83-01 (Tgf-β Inhibitor), and CHIR99021 (GSK3 Inhibitor) (YAC) small molecules to primary hepatocytes is known to convert terminally differentiated cells to bipotent liver progenitors. Tgf-β suppression and Wnt activation also induce intestinal stem cell proliferation. We therefore hypothesized that addition of YAC inhibitors might increase the proliferation and size of cultured OU to increase TESI yields for future human therapies.
Methods:
Small intestine from 2-week old C57/BL6 mice was harvested to generate OU that were maintained on reduced-factor Matrigel for 5 days (n=6). OU were cultured in DMEM supplemented with 10% fetal bovine serum, non-essential amino acids, and antibiotics in either the presence or absence of YAC small molecules refreshed every two days. Eight random fields in each well were imaged to assess the number and average area of OU (ImageJ software) and evaluated by ANOVA (GraphPad Prism 7). OU were collected on Day 5 either for whole mount or in agarose embedded in paraffin, then sectioned at 8 µm thickness. OU were co-stained for E-cadherin (epithelial marker) and Ki-67 (proliferation marker) for immunofluorescence. Paraffin-embedded sections were imaged with fluorescence microscopy and whole mount fixed OU were imaged with confocal microscopy. For each condition, OU containing a lumen were randomly imaged. The ratio of Ki-67 positive cells and total cells stained with E-Cadherin was measured and evaluated by paired t-test.
Results:
On day 5, OU treated with YAC were significantly larger than OU cultured in control media (49928 µm2 vs 9529 µm2, p=0.0036) (Figure 1A and 1B). While there was initially a significant increase in number of OU in each well with YAC application after day 1 (170.6 vs 264.5, p<0.0001), there was no significant difference in OU number on Day 5 (p=0.07). There was an increase in intestinal cell proliferation as seen by the significantly higher ratio of Ki-67 positive cells over total cells stained with E-cadherin in our YAC group (0.488 vs 0.124, p=0.0248) (Figure 1C).
Conclusion:
While there is not an increase in the overall number of OU, the addition of YAC small molecules in culture increases both the size and proliferation index of intestine organoid units. Ongoing investigation will determine if this approach increases the yield of resulting tissue-engineered small intestine growth.
