Expression of NR5A2, NUP153, HNF4A, USP15 and FNDC3B is consistent with their use as novel biomarkers for bovine mammary stem/progenitor cells.

Mammary stem cells (MaSC) are essential for growth and maintenance of mammary epithelium. Previous studies have utilized morphological characteristics or retention of bromodeoxyuridine (BrdU) label to identify MaSC and progenitor cells, these approaches may not be feasible or may not identify all resident stem cells. Alternatively, these special cells may be identified by assessing protein and mRNA expression of appropriate markers. The focus of this study was to assess the staining patterns and in situ quantification of novel candidate markers for bovine MaSC/progenitor cells. The candidate markers for MaSC/progenitor cells for immunohistochemical analysis were: NR5A2, NUP153, HNF4A, USP15 and FNDC3B and for in situ transcripts quantification were HNF4A and NUP153. We also evaluated protein expression pattern of presumptive MaSC markers known from the literature namely, ALDH1, MSI1 and Notch3. We found that NR5A2, NUP153, HNF4A and USP15-labeled cells represented 2.5-6% of epithelial cells prepubertally and were distributed in a fashion consistent with the location and abundance of MaSC/progenitor cells. A transient increase (10-37%) in expression of these markers was observed at peak lactation. FNDC3B was localized mainly in the nucleus prepubertally and in the cytoplasm of myoepithelial cells and nuclei of a limited number of alveolar cells during lactation. Abundant expression (~ 48%) and luminal localization of ALDH1 precludes its use as a bovine MaSC marker but may include transamplifying progenitor cells. MSI1 staining was consistent with MaSC localization. Onset of lumen formation in mammary ducts of prepubertal gland was associated with Notch 3 expression in the apical surface of luminal cells. RNAscope analysis of HNF4A and NUP153 transcripts in calf mammary gland showed very low copy numbers in a few epithelial cells, supporting the idea that these markers are expressed by fewer cells of epithelial origin. This study suggests that NR5A2, NUP153, HNF4A, USP15 and FNDC3B are likely markers for bovine MaSC/progenitor cells. Quantification of RNA transcripts of HNF4A and NUP153 in bovine MEC as potential MaSC markers are novel. Further studies to correlate protein expression of these markers with their transcripts level using single cell analysis in larger samples in lactating cow at different physiological stages are warranted.