Identification and characterization of presumptive bovine mammary stem cells
Choudhary, Ratan Kumar
Capuco, Anthony V
Mather, Ian H
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An understanding of the characteristics and regulation of mammary stem cells (MaSCs) is needed to gain insight into normal gland development and carcinogenesis. Previous profiling of MaSCs relied upon immunophenotypic selection of enzymatically dispersed cells by flow cytometry. However, these approaches involved the selection of cells that are removed from their tissue location and cellular microenvironment. In this study, I have utilized an alternative approach called laser microdissection, to excise putative MaSCs, based upon their ability to retain bromodeoxyuridine labeled DNA for an extended period, and control cells from their in situ locations in prepubertal bovine mammary cryosections. First, I established a protocol to immunostain putative MaSCs in tissue cryosections and isolate RNA of high quality. Next, I excised putative MaSCs and control cells from immunostained cryosections using laser microdissection. Global gene expression analysis by microarray provided evidence that MaSCs were located in the basal epithelium and progenitor cells located in suprabasal layers. A number of genes that were up-regulated in MaSCs and progenitor cells were identified and these are potential biomarkers. Analysis of the expression pattern of four genes (NR5A2, NUP153, HNF4A and FNDC3B) by immunohistochemistry showed that the protein expression profile was consistent with microarray data. Detailed immunohistochemical analyses of NR5A2, NUP153, HNF4A and FNDC3B in calf and cows (at various stages of lactation) revealed that their frequency and distribution were consistent with stem/progenitor cell characteristics. Finally, I attempted to manipulate stem/progenitor cells number using cultures of primary mammary epithelial cells. Expansion of stem/progenitor cell is a prerequisite for stem cell therapeutics and facilitates stem cell research. The effect of xanthosine on bovine mammary epithelial cells (MEC) was evaluated. The result of this study showed that xanthosine treatment increased cell proliferation, promoted symmetric cell division and increased expression of telomerase and a novel stem cell marker (FNDC3B). Together, these studies identified novel, potential markers for MaSCs and progenitor cells, and supported the ability of xanthosine to increase stem/progenitor cell number.