Of Microenvironments and Mammary Stem Cells

In most adult tissues there reside pools of stem and progenitor cells inside specialized microenvironments referred to as niches. The niche protects the stem cells from inappropriate expansion and directs their critical functions. Thus guided, stem cells are able to maintain tissue homeostasis throughout the ebb and flow of metabolic and physical demands encountered over a lifetime. Indeed, a pool of stem cells maintains mammary gland structure throughout development, and responds to the physiological demands associated with pregnancy. This review discusses how stem cells were identified in both human and mouse mammary glands; each requiring different techniques that were determined by differing biological needs and ethical constraints. These studies together create a robust portrait of mammary gland biology and identify the location of the stem cell niche, elucidate a developmental hierarchy, and suggest how the niche might be manipulated for therapeutic benefit.     

Is CD133 a marker of metastatic colon cancer stem cells?

The concept of the so-called cancer stem cell (CSC) holds that only a minority of cells within a tumor have the ability to generate a new tumor. Over the last decade, a large body of literature has implicated the protein CD133 as a marker of organ-specific adult stem cells and in some cancers as a bona fide CSC marker. In this issue of the JCI, Shmelkov et al. challenge the view that CD133 is a marker of CSCs in colon cancer (see the related article beginning on page 2111). CD133 was thought previously to have a very restricted distribution within tissues; the authors have used genetic knock-in models to demonstrate that CD133 in fact is expressed on a wide range of differentiated epithelial cells in adult mouse tissues and on spontaneous primary colon tumors in mice. In primary human colon tumors, all of the epithelial cells also expressed CD133, whereas metastatic colon cancers isolated from liver had distinct CD133+ and CD133- epithelial populations. Intriguingly, the authors demonstrate that the CD133+ and CD133- populations were equally capable of tumor initiation in xenografts. In light of these new findings, the popular notion that CD133 is a marker of colon CSCs may need to be revised.     

Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells

Loss of organization is a principle feature of cancers; therefore it is important to understand how normal adult multilineage tissues, such as bilayered secretory epithelia, establish and maintain their architectures. The self-organization process that drives heterogeneous mixtures of cells to form organized tissues is well studied in embryology and with mammalian cell lines that were abnormal or engineered. Here we used a micropatterning approach that confined cells to a cylindrical geometry combined with an algorithm to quantify changes of cellular distribution over time to measure the ability of different cell types to self-organize relative to each other. Using normal human mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, we demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells.     

Aphasia, family history, and the longitudinal course of senile dementia of the Alzheimer type

Aphasia, was present in a majority of subjects in a longitudinal study of 43 subjects with senile dementia of the Alzheimer type. Aphasic subjects had a more rapidly progressive course but a lower prevalence of familial cases than the study group, other study groups, or the nonaphasic subjects. Conversely, the lack of aphasia was associated with a higher prevalence of familial cases and a slower rate of progression. It is concluded that senile dementia of the Alzheimer type is a heterogeneous disorder in which the presence of aphasia early in the course signifies a nonfamilial, rapidly progressive variety of illness.     

Agraphia in dementia of the Alzheimer type.

This study describes graphic errors made in writing a simple sentence in 368 healthy older adults and individuals in different stages of dementia of the Alzheimer type. Errors of agraphia were present in both healthy and demented people and, in general, increased with the severity of dementia. The errors of agraphia were not correlated with measures of aphasia or psychometric measures of language and motor performance. Writing skill may represent procedural memory, and agraphia errors indicate alterations in long-term memory in dementia of the Alzheimer type.     

Birth order and maternal age effect in dementia of the Alzheimer type

Birth order and maternal age were unrelated to dementia of the Alzheimer type (DAT) in a study of 42 probands with clinically diagnosed DAT and 42 age-matched control subjects. Mean birth order in both groups did not differ significantly from the general population. The mean maternal age for the DAT probands was neither significantly different from that for the controls nor from that for the 1920 U.S. Caucasian population. Since the diagnostic criteria for the DAT group were only clinical, an additional 14 probands with DAT confirmed by autopsy were studied. Mean maternal age did not differ significantly in this group from the controls, the general population, or the clinically diagnosed DAT group. It was concluded that maternal age and birth order bear no special relationship to DAT in this sample.     

A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity

Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue–ECM boundary, rather than by differential homo- and heterotypic energies of cell–cell interaction. Surprisingly, interactions with the tissue–ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell–cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell–cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell–ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.Self-organization is a process that contributes to pattern formation and repair at all scales of biological complexity. At the tissue scale, defining robust strategies of self-organization is critical for engineering functional tissues, as well as for understanding development and the breakdown of tissue structure during diseases such as cancer (1). During development, two or more populations of motile cells can self-organize into spatially ordered tissues by a process referred to as cell sorting (2–4). The outcome of cell sorting can be rationalized using physical models that invoke cell-type-specific differences in interfacial energies. Interfacial energies arise through the action of a contractile cell cortex coupled to adhesion molecules (e.g., cadherins) that link the cortices of neighboring cells and signal to modulate cortical tension at specific cellular interfaces (5). In general, the organization of a tissue after cell sorting corresponds to a configuration that maximizes the formation of the most energetically favorable (hereafter referred to as most cell–cell cohesive)^† cellular interfaces (6). For example, with an intermediate level of heterotypic cell–cell cohesion the most self-cohesive cell type is typically found in the tissue core, with the less cohesive cell type spread around the tissue surface (Fig. 1A). In order for self-organization to proceed robustly by this strategy, a tissue requires and must maintain a clearly delineated hierarchy of homo- and heterotypic cell–cell cohesive interactions.Open in a separate windowFig. 1.A self-generated and binary adhesive interaction directs cell positioning in the mammary epithelium. (A) Self-organization of two initially disordered populations of cells (Center) into spatially ordered tissues. In the mammary gland, the correct architecture (Right) can go on to polarize and form a lumen. (B) Self-organization of fourth-passage primary human mammary epithelial cells in agarose (Left) and Matrigel (Right) after 24 h. In Matrigel, the reconstituted microtissue can also polarize and form a lumen over an additional 72 h (MEP, red, keratin-14/K14; LEP, green, keratin-19/K19; blue, DAPI/nuclei). (C) Experiments as in B but with MEP and LEP stained before self-organization with CellTracker Red (CTR) and CellTracker Green (CTG), respectively. (Insets) Average intensity profiles under each condition (n = 30). (D) Frequency of indicated tissue architectures for experiments in C (n > 235). (E) Representative images and average intensity plots of CellTracker-labeled MEP and LEP self-organized in Col1-functionalized agarose (n = 30) and unfunctionalized PDMS microwells (n = 20). (F) Conceptual model for self-organization by a self-generated adhesive interaction at the tissue–ECM boundary. (Inset) An image of MEP on an unfunctionalized PDMS surface (dotted line, PDMS; yellow, fibronectin-1; red, actin; blue, nuclei). (G) Representative images of cell doublets and XZ sections of single cells after 4 h on Matrigel-coated substrate (green, CTG; red, CTR; purple, QD605). (H) Distribution of measured contact angles at all interfaces (n > 42). (I) Representative images of aggregates of homogeneous MEP and LEP after 12 h in agarose wells. (J) Aggregates prepared as in G but subsequently transferred to Matrigel-coated glass for 12 h (green, K19; red, K14). Error bars are SD. (Scale bars, 10 µm.)Regulated differences in cell–cell cohesion are also thought to contribute to the self-organization and repair of adult human secretory organs such as the mammary gland (7, 8). The mammary gland, along with the prostate, salivary, lacrimal, and sweat glands, has an architecture comprising two concentrically arranged epithelial cell types as shown in Fig. 1A. However, the cells in the mammary gland dynamically regulate their cohesivity and motility to serve specialized roles at different locations and at different times. For example, the inner luminal (LEP) and outer myoepithelial (MEP) cells are known to undergo physical and chemical changes throughout development, menstrual cycles, pregnancy, involution, and the early stages of malignant disease. However, experiments using the mouse as a model system indicate that at the terminal end bud, where the lumen is filled and cell motility and rearrangements are elevated, cell positioning is rarely lost (9, 10). Moreover, deletion of key cell–cell adhesion proteins such as E- and P-cadherin has no gross effect on MEP or LEP cell positioning in the developing mouse mammary gland (11, 12). This is surprising given the established role of cadherins in guiding cell positioning through cell sorting. How self-organization remains robust to such severe changes to cell–cell cohesion remains unclear.Adult tissues also comprise populations of cells that can be heterogeneous in their molecular and physical properties (13), and the mammary gland is a prototypical example of a heterogeneous tissue, possessing considerable spatial and temporal variability within both the inner luminal and outer myoepithelial populations. For example, neighboring cells in healthy tissue can differ markedly with respect to their expression of adhesion molecules, cytoskeletal proteins, hormone receptors, and the activation of specific signaling pathways (14–17). Such heterogeneity can affect the distribution of cell–cell cohesive properties among different cell types, thus confounding the ordered hierarchy of interactions necessary to drive self-organization robustly (6). Nevertheless, normal levels of tissue heterogeneity do not affect cell positioning in the gland. Even when heterogeneity in cell–cell cohesion is artificially elevated by mosaic deletion of E-cadherin, LEP and MEP retain their relative positions efficiently (18). How self-organization remains robust among these and other heterogeneous populations of cells is poorly understood.The robustness exhibited by the mammary gland during self-organization could derive from a variety of mechanisms, including the action of intercellular regulatory networks or microenvironmental cues that fine-tune cell–cell cohesion. Here, we investigate the hypothesis that spatially restricted interfacial interactions unique to the tissue–ECM boundary are sufficient to direct robust self-organization, even among heterogeneous or changing populations of cells. To test this hypothesis, we reconstitute the self-organization of the mammary and prostate glands in vitro from aggregates of primary human cells. We then estimate the hierarchy of cell–cell and cell–ECM cohesive energies in the mammary gland to reveal that only the MEP, and not the LEP cells, adhere and spread onto the tissue–ECM boundary. Using mathematical modeling and cell-type-specific knockdown of key adhesion proteins, we show that binary (i.e., MEP adhere and LEP do not) cell–ECM cohesion dominates self-organization and is robust to changes to the hierarchy of cell–cell cohesive interactions. Our results provide a conceptual framework for understanding robust tissue formation in vivo and in vitro but also suggest several potential mechanisms through which tissue structure might break down during the progression of malignant disease.