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Expression of NR5A2, NUP153, HNF4A, USP15 and FNDC3B is consistent with their use as novel biomarkers for bovine mammary stem/progenitor cells
Journal of Molecular Histology ( IF 3.2 ) Pub Date : 2021-01-05 , DOI: 10.1007/s10735-020-09948-8
Ratan K Choudhary 1, 2 , Anthony V Capuco 1, 3
Affiliation  

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.



中文翻译:

NR5A2、NUP153、HNF4A、USP15 和 FNDC3B 的表达与其作为牛乳腺干/祖细胞的新型生物标志物的用途一致

乳腺干细胞(MaSC)对于乳腺上皮的生长和维持至关重要。先前的研究利用形态特征或保留溴脱氧尿苷 (BrdU) 标记来识别 MaSC 和祖细胞,这些方法可能不可行或可能无法识别所有驻留干细胞。或者,可以通过评估适当标记的蛋白质和 mRNA 表达来鉴定这些特殊细胞。本研究的重点是评估牛 MaSC/祖细胞新型候选标记物的染色模式和原位定量。用于免疫组织化学分析的 MaSC/祖细胞的候选标记物是:NR5A2、NUP153、HNF4A、USP15 和 FNDC3B,用于原位转录物定量的候选标记物是 HNF4A 和 NUP153。我们还评估了文献中已知的假定 MaSC 标记的蛋白质表达模式,即 ALDH1、MSI1 和 Notch3。我们发现NR5A2、NUP153、HNF4A和USP15标记的细胞占青春期前上皮细胞的2.5-6%,并且其分布方式与MaSC/祖细胞的位置和丰度一致。在哺乳高峰期观察到这些标记物的表达短暂增加(10-37%)。FNDC3B 在青春期前主要定位于细胞核,在哺乳期间定位于肌上皮细胞的细胞质和有限数量的肺泡细胞的细胞核。ALDH1 的丰富表达 (~ 48%) 和管腔定位使其不能用作牛 MaSC 标记,但可能包括转扩增祖细胞。MSI1 染色与 MaSC 定位一致。青春期前腺体乳腺导管管腔形成的开始与管腔细胞顶端表面的Notch 3表达相关。对小牛乳腺中 HNF4A 和 NUP153 转录本的 RNAscope 分析显示,少数上皮细胞中的拷贝数非常低,这支持了这些标记物由较少的上皮来源细胞表达的观点。这项研究表明 NR5A2、NUP153、HNF4A、USP15 和 FNDC3B 可能是牛 MaSC/祖细胞的标记。将牛 MEC 中 HNF4A 和 NUP153 的 RNA 转录物定量作为潜在的 MaSC 标记是新颖的。需要进一步研究,利用不同生理阶段泌乳牛的较大样本中的单细胞分析,将这些标记物的蛋白质表达与其转录物水平相关联。

更新日期:2021-01-05
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