ABSTRACT
AIMS: To investigate the growth and differentiation of intestinal stem cells on a novel tubular scaffold in vitro and in vivo. MATERIALS & METHODS: Intestinal progenitor cells from mice or humans were cultured with myofibroblasts, macrophages and/or bacteria, and evaluated in mice via omental implantation. Mucosal regeneration was evaluated in dogs after rectal mucosectomy followed by scaffold implantation. RESULTS: Intestinal progenitor cells differentiated into crypt-villi structures on the scaffold. Differentiation and scaffold coverage was enhanced by coculture with myofibroblasts, macrophages and probiotic bacteria, while the implanted scaffolds enhanced mucosal regeneration in the dog rectum. CONCLUSION: Intestinal stem cell growth and differentiation on a novel tubular scaffold is enhanced through addition of cellular and microbial components, as validated in mice and dogs.
Subject(s)
Cell Differentiation , Intestine, Small/cytology , Stem Cells/cytology , Tissue Scaffolds/chemistry , Animals , Blood Vessels/pathology , Cell Proliferation , Coculture Techniques , Disease Models, Animal , Dogs , Escherichia coli/physiology , Inflammation/pathology , Lactic Acid/chemistry , Lactobacillus/physiology , Mice, Inbred C57BL , Microvilli/ultrastructure , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Stem Cell Niche , Stem Cell TransplantationABSTRACT
In the intestine, bacterial components activate innate responses that protect the host. We hypothesize that bacterial components reduce Interleukin-8 (IL-8) production in intestinal epithelial cells stimulated by flagellin via the Toll-like receptor (TLR) signaling pathway. Caco-2 cells were pretreated with various doses of lipopolysaccharide (LPS), lipoteichoic acid (LTA), or low-dose flagellin (LDFL) for 24h. Cells were then treated with flagellin (FL) 500 ng/ml (HDFL) for another 48 h. IL-8 production was measured in the cell culture medium by ELISA. Eighty-four genes in the TLR pathway were evaluated by RT Profiler PCR Array. Pathway Studio 8.0 software was used for altered pathway analysis. HDFL induced IL-8 production by 19-fold (p<0.01). Pretreatment with LDFL at 20, 10 or 1 ng/ml reduced HDFL-induced IL-8 production by 61%, 52% and 40%, respectively (p<0.05). LPS at 50 µg/ml decreased HDFL-induced IL-8 production by 38% (p<0.05). HDFL up-regulated CXCL10, IL1B, IL-8, IRAK2, NF-κB1 and I-κB (all p<0.05). Pathway Studio analysis showed that HDFL induced cell processes including inflammation, cell death and apoptosis. Pretreatment with LDFL at 10 ng/ml down-regulated FADD, FOS, MAP4K4, MyD88, TLR2, TLR3 and TNFERSF1A compared to HDFL (all p<0.05). These down-regulated genes are integral for numerous cell functions including inflammatory response, cell death, apoptosis and infection. These results demonstrate that LPS and LDFL provoke tolerance to HDFL-induced IL-8 production. This tolerance effect was accompanied by a complex interaction of multiple genes related to inflammatory as well as other responses in the TLR pathway rather than a single gene alteration.
