Identification of the mammary epithelial stem cell has been a “source of much contention” . Methodologies utilized for the identification include mammosphere culture, fluorescence-activated cell sorting, and recapitulation of the mammary gland by single cells in vivo. “Since metaplasia often involves the transformation of undifferentiated stem or progenitor cells…” , metaplastic ability may be another attribute of these cells.
While no single marker can be considered to be cell-type specific, the preponderance of evidence presented in this paper including cellular phenotype, colorimetric reactions and multiple immunostains suggest that the K-HME cells are multipotent. Explant culture conditions select cells that are multipotent. Multipotency has been demonstrated for explant cultures of the hair follicle, bronchiole and intestine [10–14]. Sieber-Blum and colleagues showed that cells from bulge explants of whiskers of transgenic mice are pluripotent, differentiating into neurons, smooth muscle, Schwann cell, melanocytes, and chondrocytes . Using Wnt10cre/R26R double transgenic mice, they were able to trace these cells to the neural crest. A subsequent study by Yu et al. confirmed differentiation into neurons, muscle cells, endothelial cell, adipocytes and osteoblasts . The cell line developed by Delgado and colleagues from bronchiole explants co-express differentiation markers for multiple cell types of the lung and give rise to all lung epithelial lineages . Intestinal explant cultures or “organoids” are multi-cellular aggregates of intestinal mucosal progenitors and putative mucosal stem cells, which have been seeded onto scaffolds in tissue engineering experiments to create neointestines [13, 14]. The cells most competent to emerge from tissue explant cultures would appear to be the basal cells, which display the highest level of potency . Similarly, the epithelial cells described herein established by outgrowth from explant culture are basal and retain multipotency. Both Delgado et al. and Yu et al. suggest that their isolated cells resemble multipotent embryonic progenitors either in terms multi-lineage differentiation and/or expression of NANOG and OCT4. Human lung stem cells isolated by Wang and colleagues express NANOG, OCT3/4, SOX2 and KLF4 . Indeed, expression of OCT4 and NANOG has been reported in rare cells within adult tissues including bone marrow, epidermis, bronchial epithelium, myocardium, pancreas and testes . Likewise a subpopulation of K-HME cells express OCT4 and NANOG. K-HME cells also display multiple nucleoli, a characteristic of human embryonic stem cells .
A recent publication from the laboratory of Tlsty and colleagues reports findings similar to the ones presented in this manuscript . The cells utilized for their studies were isolated by a completely different method, i.e., the selection by flow cytometry of cells from reduction mammoplasties that, after lineage depletion, are CD73 positive and CD90 negative. These cells are also pluripotent and express a number of genes reported to confer multi-and pluipotency at levels comparable to embryonic stem cells. Although there are a number of similarities to the K-HMEs there are also some differences, e.g., their cells are EpCAM positive, and differentiation was effected by the addition of growth factors and supplements to the media. These differences notwithstanding, the fact that two independent laboratories using different methods have identified pluripotent, plastic cells in the breast lends credence to this discovery.
The epithelial cells described herein are metaplastic. They express basal cytokeratins 5 and 14, which are the hallmarks of the basal cells of stratified squamous epithelia , and myoepithelial cells/basal cells of the normal breast. However, a subset of luminal cells in the terminal ducts also express cytokeratin 5 . In the mouse mammary gland, the basal cell fraction is enriched in mammary stem cells . The expression of the luminal cytokeratins 8 and 18, and of vimentin in WIT-P media is of interest. WIT-P media in contrast to MEGM contains all-trans retinoic acid (ATRA), which has been shown to significantly increase the expression of cytokeratins 8, 18, 19, vimentin and ICAM-1 in oral gingival cells in vitro 42]. It also increases expression of these cytokeratins in T47D breast cancer cells . Ince, Weinberg and colleagues, selected primary breast epithelial cells by their growth in WIT-P media and transfected them with hTERT, SV-40 LT/st and H-ras-v12. The xenograft tumors formed from these cells expressed cytokeratins 8 and 18 and resembled human invasive ductal carcinoma . Those cells selected by their growth in MEGM resulted in tumors with squamous differentiation that lacked CK8/18 expression. p63, which is routinely used as a marker of myoepithelial cells, is strongly expressed by the K-HME cells. However, it is also a stem cell marker in the epidermis and limbal epithelium . In p63-null mice, the epithelium fails to stratify, and mammary buds or other epidermal appendages do not form . Pellegrini and colleagues have argued that the phenotype of p63-null mice should be ascribed to a failure to maintain the stem cell compartment. This would suggest that p63 marks the stem cells of the epidermal appendages, which includes the mammary glands, as well as the epidermis and limbic epithelium. It is entirely possible that the K-HME cells are the p63, CK14 and nestin positive cells identified by Li et al. in the basal/myoepithelial layer of the mammary gland . K-HME cells are EpCAM negative and CD49f positive by FACS analysis, an immunophenotype ascribed by Lim et al. to the mammary stem cell enriched population .
The differentiation of human breast cells obtained from outgrowth of organoids into squames is well described . The ability of these basal cells to form “relatively large spherical structures with a central core of squamous metaplasia” on basement membrane has also been noted [49, 50]. Squamous differentiation of cells isolated from reduction mammoplasty has more recently been reported [51, 52]. Both nasal airway stem cells and tracheal airway stem cells form spheres of squamous cells “akin to squamous cell metaplasia” when grown on Matrigel® . It should be noted that the squamous differentiation observed in our study is contextual: In the middle of Matrigel®, the phenotype most closely represents a squamous carcinoma of the skin. This keratin pearl-like structure is the form assumed by squamous metaplasia in the breast both in benign (e.g. adenomyoepitheliomas) or malignant (e.g., metaplastic squamous cell carcinoma) lesions. It is also observed in squamous cell carcinoma of the lung, esophagus, anus and even in a minority of tibial adamantinomas . This suggests a commonality in the pathophysiology of the metaplasia, that is, that basal/stem cells on becoming surrounded on all sides by basement membrane/stroma form keratin pearl structures. The fact that this is observed in normal cells raises the possibility that metaplasia is a property of all epithelia, which is kept in check by the normal microenvironment and tissue polarity. In a study conducted by Miyoshi and colleagues using transgenic mice, stabilization of β-catenin expression through MMTV-Cre-induced deletion of exon 3 results in reversion to epidermis and squamous metaplasia in the mammary tumors that develop therein . This squamous metaplasia resembles that seen in the Matrigel® sandwich cultures in that there is a cyst-like/nodular structure with keratin in the middle encircled by a stratified epithelium. These investigators suggest that the differentiation of the mammary gland as a secretory epithelium requires suppression of β-catenin signaling, and absent this repression the phenotype reverts to epidermis . In other words, the default genetic program for epithelial cells in the breast may be epidermis and their differentiation into a gland requires, at a minimum, the repression of the default program, if not a concomitant activation of a program that results in gland formation.
How can differentiation into these various cell types be explained? Boecker and colleagues recently published a study of salivary gland tumors of the breast and histologically similar tumors of the salivary and lacrimal glands . They utilized triple immunofluorescence to trace the lineage of cells within these tumors. The results of their study led them to hypothesize that there are K5/K14/p63-positive progenitor cells within these neoplasms that give rise to glandular epithelial cells, myoepithelial cells, as well as the squamous and mesenchymal cells. The K-HME cells may be the progenitor cells hypothesized by Boecker et al.
Eric Neilson has suggested that terminal differentiation rather than being an end point is a lay-over point: “…terminal differentiation is really just an evolutionary pause maintained by signaling events, transcription factors, and genomic setting” . Neilson and his colleague, Michael Zeisberg, have proposed that epithelial plasticity is comprised of two processes: Metaplasia (transdifferentiation) and epithelial-mesenchymal transition (EMT) [57, 58]. EMT can further be divided into three types [58, 59]. Type 1 EMT functions in early embryogenesis when it is involved in gastrulation and neural crest migration. Type 2 EMT is the formation of fibroblasts from secondary epithelial cells or endothelial cells. Type 3 EMT facilitates the metastasis of epithelial cells in a process that includes the loss of intercellular connections, migration and the establishment of residence in a secondary location.
Metaplasia is often composed of the tissue type normally derived from the neighboring region of the embryo [36, 60]. A dividing line forms between these two regions at a point where an inducer is at its threshold concentration . If a stem cell originally residing in this region and now in one of the resulting adult tissues retains bidirectional tissue potential, an inciting event after birth, e.g., infection, wounding, tissue regeneration, could tip the balance resulting in an homeotic transformation. The cells that eventually form the breast begin their life as ectoderm, which borders the neural crest. Neural crest cells form cartilage, bone, nerve and smooth muscle in face and cranium; as well as the peripheral nervous system and a number of neuroendocrine cell types. Pleomorphic adenomas, tumors that also display areas of bone and cartilage formation, are hypothesized to have a contribution from neural crest cells based upon the expression of GFAP . Human genetic disorders may provide an additional clue. Mutation of p63 is responsible for Limb-mammary syndrome (OMIM #603543), the features of which include hypoplasia/aplasia of the mammary gland and cleft palate. That the phenotype is manifest in tissues derived from the ectoderm and neural crest suggests that the mutation was present in a progenitor of both lineages. Are the K-HMEs just such a cell? If so, the observed phenotypic plasticity observed in the K-HME cells may be more akin to Type 1 rather than Type 3 EMT.