Abstract
Although small intestinal epithelial stem cells form crypts when using intestinal culture conditions, colon stem cells usually form colonospheres. Colon mesenchymal cell feeder layers can stimulate colon crypts to form organoids and produce crypts. We have investigated whether conditioned medium from colon mesenchymal cells can also stimulate colonosphere and organoid cryptogenesis. We prepared conditioned medium (CM) from WEHI-YH2 cells (mouse colon myofibroblasts); the CM stimulated both colonosphere formation and organoid cryptogenesis in vitro. The colon organoid-stimulating factors in WEHI-YH2 CM are inactivated by heating and trypsin digestion and proteins can be concentrated by ultrafiltration. Both the colonosphere- and organoid cryptogenesis- stimulatory effects of the CM are independent of canonical Wnt and Notch signaling. In contrast, bone morphogenetic protein 4 (BMP4) abolishes colonosphere formation and organoid cryptogenesis. The Transforming Growth Factor beta (TGFβ) Type I receptor kinase inhibitor (A83-01) stimulates colonosphere formation, whereas the Epidermal Growth Factor receptor (EGFR) kinase inhibitor (AG1478) reduces the formation of colonospheres, but in the presence of EGF, a “just-right” concentration of AG1478 increases colon organoid cryptogenesis.
Highlights
Colon epithelium is composed of a continuous sheet of epithelial cells folded into glandular crypts
We investigate the activity of the conditioned medium produced by the WEHI-YH2 cells on colonosphere and colon crypt formation in vitro (Fig 1)
We have shown previously that both YH2 conditioned medium (YH2CM) and WEHI-YH2 feeder cells stimulates colon crypt formation in colon crypt cultures with new crypts formed from colonospheres[29]
Summary
Colon epithelium is composed of a continuous sheet of epithelial cells folded into glandular crypts. The maintenance of crypt cells is regulated by stem cells at the bottom of crypts [1]. The stem cells produce new cells within a crypt, they are responsible for generating new crypts. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), has been identified as an epithelial stem cell marker in the small intestine and the colon [2]. At the bottom of the crypt LGR5+ stem cells are self-renewing [2] and are capable of producing.
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