Expression of Putative Stem Cell Marker, Hepatocyte Nuclear Factor 4 Alpha, in Mammary Gland of Water Buffalo

Abstract

Buffaloes account for more than 56% of total milk production in India. Cyclic remodeling of mammary glands of human, mice, cow, sheep, and goat is determined by mammary stem cells. It is logical to assume that buffalo mammary gland will have mammary stem/progenitor cells. Thus far, no report exists on identification of buffalo mammary stem cells. Hepatocyte nuclear factor 4 alpha (HNF4A) is a candidate marker for hepatic progenitor cells and has recently been suggested as a marker of bovine mammary stem/progenitor cells. We hypothesized that ( 1 ) HNF4A identifies putative buffalo mammary stem/progenitor cells and ( 2 ) the number of HNF4A-positive cells increases during mastitis. Sixteen buffalo mammary samples were collected from a local slaughterhouse. Hematoxylin and eosin staining were performed on 5-micron thick sections and on the basis of gross examination and histomorphology of the mammary glands, physiological stages of the animals were estimated as non-lactating (n = 4), mastitis (n = 9), and prepubertal (n = 3). In total, 24048 cells were counted (5-10 microscopic fields/animal; n = 16 animals) of which, 40% cells were mammary epithelial cells (MEC) and 60% cells were the stromal cells. The percentage of MEC in non-lactating animals was higher compared to mastitic animals (47.3% vs. 37.3%), which was likely due to loss of MEC in mastitis. HNF4A staining was observed in nuclei of MEC of ducts, alveoli, and stromal cells. Basal location and low frequency of HNF4A-positive MEC (ranges from 0.4-4.5%) were consistent with stem cell characteristics. Preliminary study showed coexpression of HNF4A with MSI1 (a mammary stem cell marker in sheep), suggesting HNF4A was likely to be a putative mammary stem/progenitor cell marker in buffalo. HNF4A-positive MEC (basal and luminal; light and dark stained) tended to be higher in non-lactating than the mastitic animals (8.73 ± 1.71% vs. 4.29 ± 1.19%; P = 0.07). The first hypothesis that HNF4A identify putative mammary stem/progenitor cells was confirmed but the second hypothesis that the number of mammary stem/progenitor cells decreases during mastitis was unsupported. This is the first report outlining the expression of HNF4A and identification of putative mammary stem/progenitor cells in buffalo mammary gland.

Conserved and variable: Understanding mammary stem cells across species

“…Water buffalos are domesticated farm animals and provide very significant portions of dairy production in several parts of Asia. There is one report in water buffalos describing identification of MaSC according to the expression of Hepatocyte Nuclear Factor 4 alpha ( HNF4α ) . HNF4α is a liver stem cell transcription factor which is highly upregulated in bovine basal label‐retaining cells and therefore suggested as a potential mammary stem cells marker .…”

Evaluation of xanthosine treatment on gene expression of mammary glands in early lactating goats

“…ALDH1 is expressed in a variety of cells, including cancer stem cells (Ginestier et al 2007). NR5A2 is a pluripotency transcription factor of adult stem cell like that of OCT4 in embryonic stem cells (Heng et al 2010), and HNF4A , FNDC3B and NOTCH1 are potential candidate markers of mammary stem/progenitor cells (Dontu et al 2004; Choudhary et al 2013, 2016; Osińska et al 2014). Fatty acid transporter genes, CD36 and FABP3 , are co-regulatory genes and have been associated with mammary cell differentiation (Spitsberg et al 1995).…”

A 100-Year Review: Mammary development and lactation

“…Others have estimated the effects of prepubertal nutrition (Daniels et al, 2009), ovariectomy (Ellis et al, 2012), or treatment with antiestrogens (Tucker et al, 2016) on populations of putative bovine MaSC via counting of LREC. Others have estimated possible populations of bovine MaSC based on counting of cells expressing HNF4A in water buffalo (Choudhary et al, 2016) or Musashi-I in mammary tissue from sheep (Colitti and Farinacci, 2009) across different stages of development. Specific to myoepithelial cells and presumptive myoepithelial progenitor cells, Ellis and colleagues (Ballagh et al, 2008;Safayi et al, 2012;Tucker et al, 2016) have shown that smooth muscle actin or common acute lymphoblastic leukemia antigen are excellent cytoplasmic markers and transformation-related protein 63 an excellent nuclear marker for basally located myoepithelial cells and their progenitors.…”