Heterocellular molecular contacts in the mammalian stem cell niche

Adult tissue homeostasis and repair relies on prompt and appropriate intervention by tissue-specific adult stem cells (SCs). SCs have the ability to self-renew; upon appropriate stimulation, they proliferate and give rise to specialized cells. An array of environmental signals is important for maintenance of the SC pool and SC survival, behavior, and fate. Within this special microenvironment, commonly known as the stem cell niche (SCN), SC behavior and fate are regulated by soluble molecules and direct molecular contacts via adhesion molecules providing connections to local supporting cells and the extracellular matrix. Besides the extensively discussed array of soluble molecules, the expression of adhesion molecules and molecular contacts is another fundamental mechanism regulating niche occupancy and SC mobilization upon activation. Some adhesion molecules are differentially expressed and have tissue-specific consequences, likely reflecting the structural differences in niche composition and design, especially the presence or absence of a stromal counterpart. However, the distribution and identity of intercellular molecular contacts for adhesion and adhesion-mediated signaling within stromal and non-stromal SCN have not been thoroughly studied. This review highlights common details or significant differences in cell-to-cell contacts within representative stromal and non-stromal niches that could unveil new standpoints for stem cell biology and therapy.     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

Differential alteration of stem and other cell populations in ducts and lobules of TGFalpha and c-Myc transgenic mouse mammary epithelium

Genes associated with proliferation are active in stem and progenitor cells, and their over-expression can promote cancer. Two such genes, c-Myc and TGFalpha, promote morphologically dissimilar mammary tumors in transgenic mice. We investigated whether their over-expression affects population size and cell cycle activity in stem and other cell populations in non-neoplastic mammary epithelia. Results indicated that both cell population and cell cycle regulation are cell type- and microenvironment-specific. To create a tool for identifying and categorizing the five cellular phenotypes by light microscopy, we adapted previously established ultrastructural criteria. Using nulliparous MMTV-c-myc or MT-tgfalpha mice, we determined and compared the relative sizes the putative stem, progenitor and differentiated cell populations. PCNA staining was used to compare the portion of each cell population in the cell cycle. Cell population sizes were analyzed relative to: (1) their location in ducts versus lobules (microenvironment), (2) genotype, and (3) cell type. Population sizes differed significantly by genotype, depending on microenvironment (p=0.0008), by genotype, depending on cell type (p<0.0001), and by microenvironment, depending on cell type (p=0.03). The number of cycling cells was also affected by all three factors, confirming that the interplay of cell type, gene expression and three-dimensional organization are very important in tissue morphogenesis and function. We describe a structure in mammary epithelium consistent with that of a stem cell niche, and show that it is altered in MMTV-c-myc and likely altered in MT TGFalpha transgenic epithelia.     

A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level. Cinzia Cocola, Sveva Sanzone and Simonetta Astigiano have contributed equally to this work.     

Current knowledge on the multiform reconstitution of intestinal stem cell niche

The development of “mini-guts” organoid originates from the identification of Lgr5⁺ intestinal stem cells (ISCs) and circumambient signalings within their specific niche at the crypt bottom. These in vitro self-renewing “mini-guts”, also named enteroids or colonoids, undergo perpetual proliferation and regulated differentiation, which results in a high-performance, self-assembling and physiological organoid platform in diverse areas of intestinal research and therapy. The triumphant reconstitution of ISC niche in vitro also relies on Matrigel, a heterogeneous sarcoma extract. Despite the promising prospect of organoids research, their expanding applications are hampered by the canonical culture pattern, which reveals limitations such as inaccessible lumen, confine scale, batch to batch variation and low reproducibility. The tumor-origin of Matrigel also raises biosafety concerns in clinical treatment. However, the convergence of breakthroughs in cellular biology and bioengineering contribute to multiform reconstitution of the ISC niche. Herein, we review the recent advances in the microfabrication of intestinal organoids on hydrogel systems.     

Irregular distribution of mammary-derived growth inhibitor in the bovine mammary epithelium

Localization of a mammary-derived growth inhibitor (MDGI) in the bovine mammary gland was verified by light-and electron-microscopic methods. Expression of MDGI, which is known to inhibit the growth of mammary epithelial cell lines in vitro, was found to be highest in the late pregnant and in the lactating state. A combination of immunohistochemical and immunocytochemical methods with semi- and ultrathin resin sections revealed marked variations in MDGI staining. High MDGI levels were predominantly detectable in epithelial cells with large milk fat droplets. Distinct cell types that were almost free of label could be identified among bovine mammary epithelial cells that always exhibited high MDGI levels. Similar results were obtained when using a serum-free organ culture system in which MDGI was hormonally induced in cell types of comparable differentiation state. The specific occurrence of the growth inhibitor in developing alveoli and certain cell types points to the association between MDGI expression and functional differentiation in the normal mammary gland.     

From gut to glutes: The critical role of niche signals in the maintenance and renewal of adult stem cells

Stem cell behavior is tightly regulated by spatiotemporal signaling from the niche, which is a four-dimensional microenvironment that can instruct stem cells to remain quiescent, self-renew, proliferate, or differentiate. In this review, we discuss recent advances in understanding the signaling cues provided by the stem cell niche in two contrasting adult tissues, the rapidly cycling intestinal epithelium and the slowly renewing skeletal muscle. Drawing comparisons between these two systems, we discuss the effects of niche-derived growth factors and signaling molecules, metabolic cues, the extracellular matrix and biomechanical cues, and immune signals on stem cells. We also discuss the influence of the niche in defining stem cell identity and function in both normal and pathophysiologic states.     

Bidirectional signaling of mammary epithelium and stroma: implications for breast cancer--preventive actions of dietary factors

The mammary gland is composed of two major cellular compartments: a highly dynamic epithelium that undergoes cycles of proliferation, differentiation and apoptosis in response to local and endocrine signals and the underlying stroma comprised of fibroblasts, endothelial cells and adipocytes, which collectively form the mammary fat pad. Breast cancer originates from subversions of normal growth regulatory pathways in mammary epithelial cells due to genetic mutations and epigenetic modifications in tumor suppressors, oncogenes and DNA repair genes. Diet is considered a highly modifiable determinant of breast cancer risk; thus, considerable efforts are focused on understanding how certain dietary factors may promote resistance of mammary epithelial cells to growth dysregulation. The recent indications that stromal cells contribute to the maintenance of the mammary epithelial ‘niche’ and the increasing appreciation for adipose tissue as an endocrine organ with a complex secretome have led to the novel paradigm that the mammary stromal compartment is itself a relevant target of bioactive dietary factors. In this review, we address the potential influence of dietary factors on mammary epithelial-stromal bidirectional signaling to provide mechanistic insights into how dietary factors may promote early mammary epithelial differentiation to decrease adult breast cancer risk.     

Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche

Background

Hematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.

Results

By utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.

Conclusion

The current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.     

干细胞壁龛功能的研究进展

随着干细胞研究的不断深入,人们愈来愈重视干细胞在机体组织中的居住环境——壁龛(niche)对干细胞的影响。干细胞的增殖分化行为受其所处微环境的影响。干细胞壁龛通过与干细胞之间的直接和(或)间接作用影响干细胞的命运。壁龛成分——壁龛细胞、细胞外基质和来源于壁龛细胞的可溶性因子在维持干细胞的特征、调控干细胞数量等方面发挥重要作用。     

A mammary repopulating cell population characterized in mammary anlagen reveals essential mammary stroma for morphogenesis

The cells with mammary repopulating capability can achieve mammary gland morphogenesis in a suitable cellular microenvironment. Using cell surface markers of CD24, CD29 and CD49f, mouse mammary repopulating unit (MRU) has been identified in adult mammary epithelium and late embryonic mammary bud epithelium. However, embryonic MRU remains to be fully characterized at earlier mammary anlagen stage. Here we isolated discrete populations of E14.5 mouse mammary anlagen cells. Only Lin⁻CD24^medCD29⁺ cell population was predicted as E14.5 MRU by examining their capacities of forming mammosphere and repopulating cleared mammary fat pad in vivo. However, when we characterized gene expressions of this E14.5 cell population by comparing with adult mouse MRU (Lin⁻CD24⁺CD29^hi), the gene profiling of these two cell populations exhibited great differences. Real-time PCR and immunostaining assays uncovered that E14.5 Lin⁻CD24^medCD29⁺ cell population was a heterogeneous stroma-enriched cell population. Then, limiting dilutions and single-cell assays also confirmed that E14.5 Lin⁻CD24^medCD29⁺ cell population possessed low proportion of stem cells. In summary, heterogeneous Lin⁻CD24^medCD29⁺ cell population exhibited mammary repopulating ability in E14.5 mammary anlagen, implying that only suitable mammary stroma could enable mammary gland morphogenesis, which relied on the interaction between rare stem cells and microenvironment.     

The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche

Maintenance of adult stem cells is largely dependent on the balance between their self-renewal and differentiation. The Drosophila ovarian germline stem cells (GSCs) provide a powerful in vivo system for studying stem cell fate regulation. It has been shown that maintaining the GSC population involves both genetic and epigenetic mechanisms. Although the role of epigenetic regulation in this process is evident, the underlying mechanisms remain to be further explored. In this study, we find that Enoki mushroom (Enok), a Drosophila putative MYST family histone acetyltransferase controls GSC maintenance in the ovary at multiple levels. Removal or knockdown of Enok in the germline causes a GSC maintenance defect. Further studies show that the cell-autonomous role of Enok in maintaining GSCs is not dependent on the BMP/Bam pathway. Interestingly, molecular studies reveal an ectopic expression of Bruno, an RNA binding protein, in the GSCs and their differentiating daughter cells elicited by the germline Enok deficiency. Misexpression of Bruno in GSCs and their immediate descendants results in a GSC loss that can be exacerbated by incorporating one copy of enok mutant allele. These data suggest a role for Bruno in Enok-controlled GSC maintenance. In addition, we observe that Enok is required for maintaining GSCs non-autonomously. Compromised expression of enok in the niche cells impairs the niche maintenance and BMP signal output, thereby causing defective GSC maintenance. This is the first demonstration that the niche size control requires an epigenetic mechanism. Taken together, studies in this paper provide new insights into the GSC fate regulation.     

Regeneration of the amphibian intestinal epithelium under the control of stem cell niche

The epithelium of the mammalian digestive tract originates from stem cells and undergoes rapid cell-renewal throughout adulthood. It has been proposed that the microenvironment around the stem cells, called 'niche', plays an important role in epithelial cell-renewal through cell-cell and cell-extracellular matrix interactions. The amphibian intestine, which establishes epithelial cell-renewal during metamorphosis, serves as a unique and good model for studying molecular mechanisms of the stem cell niche. By using the organ culture of the Xenopus laevis intestine, we have previously shown that larval-to-adult epithelial remodeling can be organ-autonomously induced by thyroid hormone (TH) and needs interactions with the surrounding connective tissue. Thus, in this animal model, the functional analysis of TH response genes is useful for clarifying the epithelial-connective tissue interactions essential for intestinal remodeling at the molecular level. Recent progress in culture and transgenic technology now enables us to investigate functions of such TH response genes in the X. laevis intestine and sheds light on molecular aspects of the stem cell niche that are common to the mammalian intestine.     

Cilia on bovine mammary epithelium: ultrastructural observations

Ultrastructural examination of bovine mammary tissues revealed the presence of 9+0 or primary cilia protruding from surfaces of alveolar epithelial and myoepithelial cells. Cilia of epithelial cells protruded approximately 1200 nm into lumina of alveoli and arose from a basal body centriole, the associated centriole of the diplosome, and an accessory rootlet system. Cilia on epithelial cells were more frequently observed than cilia on myoepithelial cells. Occasional cilia made contact with macrophages in the alveolar lumen. The structures were more commonly found in tissues from nonlactating cows, and most were observed in the ventral portion of the mammary gland.     

The neural stem cell niche

The neural stem cell niche defines a zone in which stem cells are retained after embryonic development for the production of new cells of the nervous system. This continual supply of new neurons and glia then provides the postnatal and adult brain with an added capacity for cellular plasticity, albeit one that is restricted to a few specific zones within the brain. Critical to the maintenance of the stem cell niche are microenvironmental cues and cell-cell interactions that act to balance stem cell quiescence with proliferation and to direct neurogenesis versus gliogenesis lineage decisions. Ultimately, based on the location of the niche, stem cells of the adult brain support regeneration in the dentate gyrus of the hippocampus and the olfactory bulb through neuron replacement. Here, we provide a summary of the current understanding of the organization and control mechanisms of the neural stem cell niche.     

干细胞巢研究进展

干细胞巢即干细胞周围的微环境构成,一般包括干细胞的相邻细胞、粘附分子及基质等,但不同的干细胞有不同的巢结构。干细胞巢通过不同信号途径调控着干细胞的行为,使干细胞的自我更新和分化处于平衡状态。根据近年来有关干细胞巢的研究,本文从果蝇生殖系干细胞巢、哺乳动物造血干细胞巢、肠干细胞巢、毛囊表皮干细胞巢和神经干细胞巢等五个系统分别综述了干细胞巢的构成及其对干细胞的调节作用,探讨了干细胞巢作用于干细胞的内在机制。     

Multicellular cuddling in a stem cell niche

Haematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate in any blood cell type throughout life and thereby sustain the entire blood system. To do so, HSPCs had been shown to seed, in a multi-step process, intermediate haematopoietic niches before colonizing the adult marrow. While HSPC birth had been thoroughly characterized in the past, both in mammals and in zebrafish, how perivascular niches could host HSPCs and sustain their expansion was poorly understood. In an article published in the last issue of Cell, Tamplin et al.¹ elegantly exploited the many advantages provided by the zebrafish embryo to describe how endothelium remodeling in the perivascular niche, referred to as “cuddling,” favors HSPCs colonization and expansion.