Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERα^neg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.     

Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

A Small Molecule Inhibitor of Src Family Kinases Promotes Simple Epithelial Differentiation of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs, the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA) – induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8), which are expressed in simple epithelial cells, while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4− simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications.     

A CK19(CreERT) knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs

Cre/LoxP-mediated DNA recombination allows for gene function and cell lineage analyses during embryonic development and tissue regeneration. Here, we describe the derivation of a K19(CreERT) mouse line in which the tamoxifen-activable CreER(T) was knocked into the endogenous cytokeratin 19 locus. In the absence of tamoxifen, leaky Cre activity could be detected only in less than 1% of stomach and intestinal epithelial cells, but not in pancreatic or hepatic epithelial tissues. Tamoxifen administration in postnatal animals induced widespread DNA recombination in epithelial cells of pancreatic ducts, hepatic ducts, stomach, and intestine in a dose-dependent manner. Significantly, we found that Cre activity could be induced in the putative gut stem/progenitor cells that sustained long-term gut epithelial expression of a Cre reporter. This mouse line should therefore provide a valuable reagent for manipulating gene activity and for cell lineage marking in multiorgans during normal tissue homeostasis and regeneration.     

Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.     

The dynamics of murine mammary stem/progenitor cells

The stem/progenitor cells in the murine mammary gland are a highly dynamic population of cells that are responsible for ductal elongation in puberty, homeostasis maintenance in adult, and lobulo-alveolar genesis during pregnancy. In recent years understanding the epithelial cell hierarchy within the mammary gland is becoming particularly important as these different stem/progenitor cells were perceived to be the cells of origin for various subtypes of breast cancer. Although significant advances have been made in enrichment and isolation of stem/progenitor cells by combinations of antibodies against cell surface proteins together with flow cytometry, and in identification of stem/progenitor cells with multi-lineage differentiation and self-renewal using mammary fat pad reconstitution assay and in vivo genetic labeling technique, a clear understanding of how these different stem/progenitors are orchestrated in the mammary gland is still lacking. Here we discuss the different in vivo and in vitro methods currently available for stem/progenitor identification, their associated caveats, and a possible new hierarchy model to reconcile various putative stem/progenitor cell populations identified by different research groups.     

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

Summary The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm-Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

Localization of putative stem cells and four cell populations with different differentiation degree in mouse mammary anlagen

The precise localization of putative stem cells and other cells within the embryonic mammary gland would help to elucidate the molecular pathways that govern normal mammary development. The ultrastructural appearances and the antigen of Sca-1 were considered together as putative stem cell markers, and the antigens of cytokeratin, CD10, Muc-1 and CD34 as differentiation markers. Electron microscopy was performed to reveal the ultrastructure of cells in different site of the mammary anlagen. An immunofluorescence system was used to reveal the whole structure profile of the mammary anlagen using the anti-cytokeratin antibody to localize specific types of cell populations such as Sca-1, CD10, Muc-1 and CD34 positive cells within the anlagen, which distribute near the inside edge, distribute uniformly, distribute in the central region and distribute in the site of mesenchyme around the neck of the anlagen, respectively. We also observed under electron microscope that some pale cells like putative stem cells reported by prevenient scholars, which are mainly distributed in the Sca-1⁺ cell population near the inside edge of the anlagen, have pale-stained nucleoplasm and cytoplasm, sparse organelles clustered close to their nucleus and have a lack of rough endoplasmic reticulums and cell polarization. The results indicate that the putative stem cells are localized near the edge of the mammary anlagen; the cell populations with different differentiation degree were localized in the central part and around the edge within the anlagen.     

Cytokeratin expression in human thymus: immunohistochemical mapping

Cytokeratin expression in normal postnatal human thymus was studied immunohistochemically by using monoclonal antibodies against various cytokeratin polypeptides. An attempt was made to characterize cell populations giving rise to the cornified structures of Hassal's corpuscles. Monoclonal antibody KB-37, a marker of squamous epithelium basal cells, was applied to distinguish the earliest cells capable of undergoing squamous differentiation. Parts of the subcapsular epithelium were extensively stained with this reagent. This epithelium, like the basal layer of certain squamous epithelia, exibited a high incidence of cytokeratins 13 and 14, and pronounced expression of cytokeratin 19. Simple epithelium cytokeratins 8, 18, and 19 were present in the cortex. Scattered cells reacted with KB-37 antibody. All stellate epithelial cells in the medulla were positive for cytokeratin 19. Most of the medullar epithelial cells were positive for cytokeratins 13, 14 and 17 of complex epithelium, in contrast to the cortex, where only a few cells were positive for these cytokeratins. A significant proportion of the medullar cells was positive for KB-37 antigen. Cytokeratins 8 and 18 were expressed in single cells and in groups of cells surrounding Hassal's corpuscles. The outermost cells of these corpuscles were positive for cytokeratin 19 and KB-37. In the peripheral parts of Hassal's corpuscles, simple epithelium cytokeratins 7, 8, 18, and cytokeratins 4, 13, 14, and 17, characteristic of stratified nonkeratinizing epithelia, were coexpressed with keratinization-specific cytokeratins 10/11. The inner parts of the swirls were uniformly positive for cytokeratins was reduced.     

RARα and RARγ reciprocally control K5+ progenitor cell expansion in developing salivary glands

Understanding the mechanisms of controlled expansion and differentiation of basal progenitor cell populations during organogenesis is essential for developing targeted regenerative therapies. Since the cytokeratin 5-positive (K5⁺) basal epithelial cell population in the salivary gland is regulated by retinoic acid signaling, we interrogated how isoform-specific retinoic acid receptor (RAR) signaling impacts the K5⁺ cell population during salivary gland organogenesis to identify RAR isoform-specific mechanisms that could be exploited in future regenerative therapies. In this study, we utilized RAR isoform-specific inhibitors and agonists with murine submandibular salivary gland organ explants. We determined that RARα and RARγ have opposing effects on K5⁺ cell cycle progression and cell distribution. RARα negatively regulates K5⁺ cells in both whole organ explants and in isolated epithelial rudiments. In contrast, RARγ is necessary but not sufficient to positively maintain K5⁺ cells, as agonism of RARγ alone failed to significantly expand the population. Although retinoids are known to stimulate differentiation, K5 levels were not inversely correlated with differentiated ductal cytokeratins. Instead, RARα agonism and RARγ inhibition, corresponding with reduced K5, resulted in premature lumenization, as marked by prominin-1. With lineage tracing, we demonstrated that K5⁺ cells have the capacity to become prominin-1⁺ cells. We conclude that RARα and RARγ reciprocally control K5⁺ progenitor cells endogenously in the developing submandibular salivary epithelium, in a cell cycle-dependent manner, controlling lumenization independently of keratinizing differentiation. Based on these data, isoform-specific targeting RARα may be more effective than pan-RAR inhibitors for regenerative therapies that seek to expand the K5⁺ progenitor cell pool. Summary statement: RARα and RARγ reciprocally control K5⁺ progenitor cell proliferation and distribution in the developing submandibular salivary epithelium in a cell cycle-dependent manner while regulating lumenization independently of keratinizing differentiation.     

Essential Roles of Cyclin Y-Like 1 and Cyclin Y in Dividing Wnt-Responsive Mammary Stem/Progenitor Cells

Cyclin Y family can enhance Wnt/β-catenin signaling in mitosis. Their physiological roles in mammalian development are yet unknown. Here we show that Cyclin Y-like 1 (Ccnyl1) and Cyclin Y (Ccny) have overlapping function and are crucial for mouse embryonic development and mammary stem/progenitor cell functions. Double knockout of Ccnys results in embryonic lethality at E16.5. In pubertal development, mammary terminal end buds robustly express Ccnyl1. Depletion of Ccnys leads to reduction of Lrp6 phosphorylation, hampering β-catenin activities and abolishing mammary stem/progenitor cell expansion in vitro. In lineage tracing experiments, Ccnys-deficient mammary cells lose their competitiveness and cease to contribute to mammary development. In transplantation assays, Ccnys-deficient mammary cells fail to reconstitute, whereas constitutively active β-catenin restores their regeneration abilities. Together, our results demonstrate the physiological significance of Ccnys-mediated mitotic Wnt signaling in embryonic development and mammary stem/progenitor cells, and reveal insights in the molecular mechanisms orchestrating cell cycle progression and maintenance of stem cell properties.     

Differentiation of mouse embryonic palatal epithelium in culture: selective cytokeratin expression distinguishes between oral, medial edge and nasal epithelial cells

During normal murine palatogenesis, regional specific differentiation of the epithelium results in three cell phenotypes: nasal (ciliated pseudostratified columnar cells), oral (stratified squamous cells) and medial edge (migratory, epithelio-mesenchymally transformed cells). We have developed a defined, serum-free, culture system which supports the growth and differentiation of isolated murine embryonic palatal epithelia in vitro. Using immunofluorescence microscopy, an established panel of antibodies was used to characterise the cytokeratin intermediate filament profile of palatal epithelial sheets at a precise developmental stage, following culture in serum-free medium with and without either transforming growth factor alpha (TGF alpha) or 10% donor calf serum (DCS). The morphologically discernable oral, medial edge and nasal phenotypes exhibited distinctive cytokeratin profiles, which remained consistent for all culture conditions, and which correlated with the known differentiation states of the epithelial types. The oral epithelia stained positively for cytokeratin 19 and cytokeratins characteristic of multilayered epithelia (1, 5, 14). Nasal epithelia stained similarly but in addition expressed the simple-epithelial cytokeratin pair, 8 and 18. Medial edge epithelia also expressed cytokeratins 1, 5 and 14 but with the exception of a few isolated cells there was no staining for cytokeratins 8 and 18. Cytokeratin 19 was absent specifically from the medial edge epithelial cells: this result may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformations. By exhibiting a complexity of expression linked to differentiation state and independent of culture conditions, cytokeratins constitute useful markers of palatal epithelial differentiation in vitro as well as in vivo.     

Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells

To establish a method for efficient and relatively easy isolation of a cell population containing epithelial prostate stem cells, we developed two transgenic mouse models, K5/CFP and K18/RFP. In these models, promoters of the cytokeratin 5 (Krt5) and the cytokeratin 18 (Krt18) genes regulate cyan and red fluorescent proteins (CFP and RFP), respectively. CFP and RFP reporter protein fluorescence allows for visualization of K5⁺ and K18⁺ epithelial cells within the cellular spatial context of the prostate gland and for their direct isolation by FACS. Using these models, it is possible to test directly the stem cell properties of prostate epithelial cell populations that are positively selected based on expression of cytoplasmic proteins, K5 and K18. After validating appropriate expression of the K5/CFP and K18/RFP transgenes in the developing and adult prostate, we demonstrate that a subset of CFP-expressing prostate cells exhibits stem cell proliferation potential and differentiation capabilities. Then, using prostate cells sorted from double transgenic mice (K5/CFP + K18/RFP), we compare RNA microarrays of sorted K5⁺K18⁺ basal and K5⁻K18⁺ luminal epithelial cells, and identify genes that are differentially expressed. Several genes that are over-expressed in K5⁺ cells have previously been identified as potential stem cell markers. These results suggest that FACS isolation of prostate cells from these mice based on combining reporter gene fluorescence with expression of potential stem cell surface marker proteins will yield populations of cells enriched for stem cells to a degree that has not been attained by using cell surface markers alone.     

Expression of intermediate filament proteins in fetal and adult human lung tissues

The expression patterns of intermediate filament proteins in fetal and normal or nonpathological adult human lung tissues are described using (chain-specific) monoclonal antibodies. In early stages of development (9-10 weeks and 25 weeks of gestation) only so-called simple cytokeratins such as cytokeratins 7 (minor amounts). 8, 18 and 19 are detected in bronchial epithelial cells. At later stages of development, the cytokeratin expression patterns become more complex. The number of bronchial cells positive for cytokeratin 7 increases, but basal cells in the bronchial epithelium remain negative. These latter cells show, however, expression of cytokeratin 14 in the third trimester of gestation. Developing alveolar epithelial cells express cytokeratins 7, 8, 18 and 19. In adult human bronchial epithelium cytokeratins 4 (varying amounts), 7, 8, 13 (minor amounts), 14, 18 and 19 can be detected, with the main expression of cytokeratins 7, 8, and 18 in columnar cells and the main expression of cytokeratin 14 in basal cells. Vimentin is detected in all mesenchymal tissues. In addition, fetal lung expresses vimentin in bronchial epithelium, however, to a lesser extent with increasing age, resulting in the expression of vimentin in only few scattered bronchial cells at birth. Also in adult bronchial epithelium the expression of vimentin is noticed in part of the basal and columnar epithelial cells. Desmin filaments, present in smooth muscle cells of the lung, appear to alter their protein structure with age. In early stages of development smooth muscle cells surrounding blood vessels are partly reactive with some cytokeratin antibodies and with a polyclonal desmin antibody. At week 9-10 and week 25 of gestation a monoclonal antibody to desmin, however, is not reactive with blood vessel smooth muscle cells but is only reactive with smooth muscle cells surrounding bronchi. With increasing age the reactivity of cytokeratin antibodies with smooth muscle cells in blood vessels decreases, while the reactivity with the monoclonal desmin antibody increases. Our results show that during differentiation profound changes in the intermediate filament expression patterns occur in the different cell types of the developing lung.     

Isolation and characterization of functional mammary gland stem cells

Abstract.  Significant advances in the stem-cell biology of several tissues, including the mammary gland, have occurred over the past several years. Recent progress on stem-cell fate determination, molecular markers, signalling pathways and niche interactions in haematopoietic, neuronal and muscle tissue may provide parallel insight into the biology of mammary epithelial stem cells. Taking advantage of approaches similar to those employed to isolate and characterize haematopoietic and epidermal stem cells, we have identified a mammary epithelial cell population with several stem/progenitor cell qualities. In this article, we review some recent data on mammary epithelial stem/progenitor cells in genetically engineered mouse models. We also discuss several potential molecular markers, including stem-cell antigen-1 (Sca-1), which may be useful for both the isolation of functional mammary epithelial stem/progenitor cells and the analysis of tumour aetiology and phenotype in genetically engineered mouse models. In different transgenic mammary tumour models, Sca-1 expression levels, as well as several other putative markers of progenitors including keratin-6, possess dramatically altered expression profiles. These data suggest that the heterogeneity of mouse models of breast cancer may partially reflect the selection or expansion of different progenitors.     

Coxsackievirus and adenovirus receptor-positive cells compose the putative stem/progenitor cell niches in the marginal cell layer and parenchyma of the rat anterior pituitary

The pituitary gland is a slow generative tissue but actively responds to demands by changing homeostasis. The marginal cell layer (MCL) facing the residual lumen has long been indicated as a stem/progenitor cell niche of the pituitary. On the other hand, the coxsackievirus and adenovirus receptor (CAR), which localizes at the tight-junction of the polarized epithelium, is known to participate in the development, differentiation and regeneration of specified tissues. The present study attempts to characterize the cells lining the MCL during pituitary development by immunohistochemistry of CAR. Consequently, we found that CAR localizes in an apical surface of the single cell layer facing the oral cavity in the invaginating oral epithelium on rat embryonic day (E) 11.5. On E13.5, when this single layer constructs the MCL in the pituitary primordium Rathke’s pouch, CAR-positive cells occupied the MCL and this localization pattern of CAR was persistently maintained throughout life. Moreover, clusters of CAR-positive cells were also found in the parenchyma. CAR-positive cells were positive for stem/progenitor cell markers sex-determining region Y-box 2 (SOX2) and epithelial calcium-dependent adhesion (E-cadherin). However, prior to the postnatal growth wave, cells positive for CAR in the basolateral surface constructed multiple cell layers beneath the MCL and cell-type transition to a putative migratory cell phenotype by fading of SOX2 and E-cadherin occurred, suggesting the composition of new putative niches in the parenchyma. These data, together with our previous reports, suggest that CAR-positive cells are pituitary stem/progenitor cells and compose putative stem/progenitor cell niches in the MCL and parenchyma.