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  • Title: Multilineage differentiation of cloned HRA-19 cells in serum-free medium: a model of human colorectal epithelial differentiation.
    Author: Henderson K, Kirkland SC.
    Journal: Differentiation; 1996 Jul; 60(4):259-68. PubMed ID: 8765056.
    Abstract:
    Colorectal epithelium is composed of polarised absorptive enterocytes, mucus-producing goblet cells and enteroendocrine cells. All these cell lineages are thought to arise from multipotential stem cells located near the base of the crypt, but the mechanisms which control differentiation and commitment of cells to a particular lineage are poorly understood. We have used the human rectal adenocarcinoma cell line, HRA-19, to investigate the regulation of expression of lineage-specific markers. HRA-19 cells have multipotential characteristics, forming absorptive, mucous and endocrine cells when grown as xenografts. However, HRA-19 cells grown in vitro in culture medium containing 10% foetal calf serum show negligible expression of the differentiated phenotypes observed in vivo. These findings initially suggested that the absence of positive stimuli from extracellular matrix, stromal cells and/or soluble factors present in vivo resulted in the lack of differentiation in vitro. The subsequent demonstration of a marked inhibitory effect of foetal calf serum on differentiation provided an alternative explanation for the differences between in vivo and in vitro differentiation. In addition, the inhibition of differentiation differed widely between batches of foetal calf serum and limited the usefulness of the system for studying the regulation of differentiation. This manuscript describes the development of chemically defined culture conditions (Dulbecco's Eagles medium supplemented with insulin, transferrin and ascorbic acid) which reproducibly induced the multilineage differentiation of HRA-19 cells into absorptive, mucous and endocrine cells. Morphological characteristics and the expression of lineage-specific markers, as determined by immunocytochemistry, identified absorptive, goblet and endocrine cells in HRA-19 monolayers grown in this serum-free medium. Differentiation of cloned HRA-19 cells in to the three cell lineages proceeds in the absence of stromal cells and without exogenous extracellular matrix, although these factors may subsequently be shown to modulate the rate of cell differentiation. These chemically defined culture conditions will facilitate the study of differentiation in the HRA-19 cell line in the absence of the complex mixture of growth factors, hormones and differentiation inhibitory factor(s) present in foetal calf serum.
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