Critical intestinal cells originate from the host in enteroid-derived tissue-engineered intestine.

BACKGROUND

Enteroid-derived tissue-engineered intestine (TEI) contains intestinal subepithelial myofibroblasts (ISEMFs) and smooth muscle cells (SMCs). However, these cell types are not present in the donor enteroids. We sought to determine the origin of these cell types and to quantify their importance in TEI development.

MATERIALS AND METHODS

Crypts from pan-EGFP or LGR5-EGFP mice were used for enteroid culture and subsequent implantation for the production of TEI. TEI from pan-EGFP enteroids was labeled for smooth muscle alpha actin (SMA) to identify ISEMFs and SMCs and green fluorescent protein (GFP) to identify cells from pan-EGFP enteroids. Fluorescence in situ hybridization (FISH) for the Y chromosome was applied to TEI from male LGR5-EGFP enteroids implanted into female nonobese diabetic/severe combined immunodeficiency mice. To identify chemotactic effects of intestinal epithelium on ISEMFs, a Boyden chamber assay was performed.

RESULTS

Immunofluorescence of TEI from pan-EGFP enteroids revealed GFP-positive epithelium with surrounding SMA positivity and no colocalization of the two. FISH of TEI from male LGR5-EGFP enteroids implanted into female nonobese diabetic/severe combined immunodeficiency mice revealed that only the epithelium was Y chromosome positive. Chemotactic assays demonstrated increased ISEMF migration in the presence of enteroids (983 ± 133) compared to that in the presence of either Matrigel alone (357 ± 36) or media alone (339 ± 24; P ≤ 0.05).

CONCLUSIONS

Lack of GFP/SMA colocalization suggests that ISEMFs and SMCs are derived from host animals. This was confirmed by FISH which identified only epithelial cells as being male. All other cell types originated from host animals. The mechanism by which these cells are recruited is unknown; however, Boyden chamber assays indicate a direct chemotactic effect of intestinal epithelium on ISEMFs.