DNA damage checkpoints in stem cells, ageing and cancer

DNA damage induces cell-intrinsic checkpoints, including p53 and retinoblastoma (RB), as well as upstream regulators (exonuclease 1 (EXO1), ataxia telangiectasia mutated (ATM), ATR, p16(INK4a) and p19(ARF)) and downstream targets (p21, PUMA (p53 upregulated modulator of apoptosis) and sestrins). Clearance of damaged cells by cell-intrinsic checkpoints suppresses carcinogenesis but as a downside may impair stem cell and tissue maintenance during ageing. Modulating the activity of DNA damage checkpoints can either accelerate or decelerate tissue ageing and age-related carcinogenesis. The outcome depends on cell-intrinsic and cell-extrinsic mechanisms that regulate the clearance of damaged cells and on the molecular context in ageing tissues, including the level of DNA damage accumulation itself.     

Regulation of adult stem cell behavior by nutrient signaling

Adult stem cells play an essential role?throughout life, maintaining tissue and organ function by providing a reservoir of cells for homeostasis and repair. Maintenance and activity of adult stem cells have been the focus of numerous studies that have revealed stem cell-intrinsic factors and signals from the local microenvironment that regulate stem cell behavior. A growing body of work has provided evidence that circulating, systemic factors also contribute to the regulation of stem cell behavior in numerous tissues. We have demonstrated that Drosophila male germline stem cells (GSCs) and intestinal stem cells (ISCs) respond to changes in nutrient availability, specifically amino acids. Furthermore, we have shown that insulin signaling plays an important role in mediating the effects of changes in nutritional conditions. Notably, insulin signaling is cell-autonomously required within male GSCs for maintenance. Here we discuss our data regarding the effects and mechanisms by which changes in systemic nutritional conditions may influence the maintenance and activity of adult stem cells via insulin signaling.     

Regulation of adult stem cell behavior by nutrient signaling

Adult stem cells play an essential role throughout life, maintaining tissue and organ function by providing a reservoir of cells for homeostasis and repair. Maintenance and activity of adult stem cells have been the focus of numerous studies that have revealed stem cell-intrinsic factors and signals from the local microenvironment that regulate stem cell behavior. A growing body of work has provided evidence that circulating, systemic factors also contribute to the regulation of stem cell behavior in numerous tissues. We have demonstrated that Drosophila male germline stem cells (GSCs) and intestinal stem cells (ISCs) respond to changes in nutrient availability, specifically amino acids. Furthermore, we have shown that insulin signaling plays an important role in mediating the effects of changes in nutritional conditions. Notably, insulin signaling is cell-autonomously required within male GSCs for maintenance. Here we discuss our data regarding the effects and mechanisms by which changes in systemic nutritional conditions may influence the maintenance and activity of adult stem cells via insulin signaling.Key words: Drosophila, stem cells, nutrition, insulin, niche     

Integrin-mediated adhesion and stem-cell-niche interactions

The regulation of stem cell behavior and maintenance typically involves the integration of both intrinsic and extrinsic cues. One such external cue, integrin-mediated cell adhesion to the extracellular matrix, plays an important part in regulating stem cell function and maintenance. In particular, integrins help define and shape the microenvironment in which stem cells are found: the stem cell niche. Integrins have a diverse array of roles in this context including homing of stem cells to their niche, maintaining stem cells in the niche, developing stem-cell-niche architecture, regulating stem cell proliferation and self renewal, and finally, controlling the orientation of dividing stem cells. Because of their various roles in directing stem cell behavior, integrin-mediated adhesion and signaling in the niche have been implicated in processes that underlie cancer progression and metastasis.     

Multiple Roles of Canonical Wnt Signaling in Cell Cycle Progression and Cell Lineage Specification in Neural Development

Signaling by the canonical Wnt pathway has multiple functions in stem cells. It caneither control stem cell expansion or –as we have recently demonstrated withneural crest stem cells– influence cell lineage decisions by promoting specific fatesat the expense of others. Thus, the role of canonical Wnt in stem cells is dependenton cell-intrinsic properties that determine how a cell responds to Wnt. Themolecular basis for the functional diversity of Wnt in different stem cell typesremains to be elucidated.     

Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.     

Stem cells in the embryonic cerebral cortex: Their role in histogenesis and patterning

The cytoarchitectural simplicity of the cerebral cortex makes it an attractive system to study central nervous system (CNS) histogenesis—the process whereby diverse cells are generated in the right numbers at the appropriate place and time. Recently, multipotent stem cells have been implicated in this process, as progenitor cells for diverse types of cortical neurons and glia. Continuous analysis of stem cell clone development reveals stereotyped division patterns within their lineage trees, highly reminiscent of neural lineage trees in arthropods and Caenorhabditis elegans. Given that these division patterns play a critical part in generating diverse neural types in invertebrates, we speculate that they play a similar role in the cortex. Because stereotyped lineage trees can be observed from cells growing at clonal density, cell-intrinsic factors are likely to have a key role in stem cell behavior. Cortical stem cells also respond to environmental signals to alter the types of cells they generate, providing the means for feedback regulation on the germinal zone. Evidence is accumulating that cortical stem cells, influenced by intrinsic programs and environmental signals, actually change with development—for example, by reducing the number and types of neurons they produce. Age-related changes in the stem cell population may have a critical role in orchestrating development; whether these cells truly self-renew is a point of discussion. In summary, we propose that cortical stem cells are the focus of regulatory mechanisms central to the development of the cortical cytoarchitecture. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 162–174, 1998     

Neuronal potential and lineage determination by neural stem cells

How do neural stem cells ensure that they give rise to the right number and type of neurons at the right time? Over the past year several regulatory mechanisms have been identified, including promotion of neurogenesis by proneural bHLH genes, instruction of gliogenesis by Notch, and cell-intrinsic changes in the neurogenic capacity of stem cells in culture and in vivo.     

Written in bone: young bone makes young blood

Ageing of bone marrow haematopoietic stem cells has been mostly associated with cell-intrinsic mechanisms. New findings published in The EMBO Journal now show that reduced levels of the secreted matrix protein osteopontin in old bone marrow stroma cause ageing-associated features in HSCs. In line, old HSCs are functionally rejuvenated by interaction with protease-cleaved osteopontin fragment, and perform like young blood stem cells in vivo.     

Anchoring stem cells in the niche by cell adhesion molecules

Adult stem cells generally reside in supporting local micro environments or niches, and intimate stem cell and niche association is critical for their long-term maintenance and function. Recent studies in model organisms especially Drosophila have started to unveil the underlying mechanisms of stem anchorage in the niche at the molecular and cellular level. Two types of cell adhesion molecules are emerging as essential players: cadherin-mediated cell adhesion for keeping stem cells within stromal niches, whereas integrin-mediated cell adhesion for keeping stem cells within epidermal niches. Further understanding stem cell anchorage and release in coupling with environmental changes should provide further insights into homeostasis control in tissues that harbor stem cells.Key words: stem cell, niche, anchorage, cell adhesion, extracellular matrix, cadherin, integrinTissue-specific adult stem cells are characterized by their prolonged self-renewal ability and potentiality to differentiate into one or more types of mature cells. These unique properties make stem cells essential for maintaining tissue homeostasis throughout life. It is generally believed that all adult stem cells reside in specific microenvironments named niches, which provide physical support and produce critical signals to maintain stem cell identity and govern their behavior.^1–4 Consequently, intimate stem cell and niche association is a pre-requisite for stem cell''s long-term maintenance and function. How stem cells are kept within the niche is thus an important issue in stem cell biology. Characterization of a number of stem cell niches in model organisms has led to the classification of niches into two general types: stromal niches where stem cells have direct membrane contact with the niche cells and epidermal niches where stem cells are usually associated with the extracellular matrix (ECM), and do not directly contact any fixed stromal cells.¹ Studies in Drosophila have led to the cellular and functional verification of the stem cell niche theory^5,6 and not surprisingly, have also led to the discovery of the molecular mechanisms anchoring stem cells to the niche. Here I consider recent studies in Drosophila on types of cell adhesions used to anchor stem cells in the niches, and summarize cell adhesion molecules utilized in the most characterized niches in the mammalian tissues, and suggest that cadherin-mediated cell-to-cell adhesion and integrin-mediated cell-to-ECM adhesion are possibly two general mechanisms that function in respective stromal or epidermal niches for stem cell anchorage in diverse organisms.     

AN ICAM-1 LIKE CELL ADHESION MOLECULE IS RESPONSIBLE FOR CD34 POSITIVE HAEMOPOIETIC STEM CELLS ADHESION TO BONE-MARROW STROMA

The microenvironment in the haematopoietic organs plays an important role in regulating and sustaining differentiation and self-renewal of haematopoietic stem cells. Although crucial for stem cell maintenance and homing, the stromal cell—stem cell interactions are poorly understood. Here we show that an ICAM-like molecule is responsible for stem cell adhesion to stromal cellsin vitro. The molecule was characterized by a monoclonal antibody 3E₁₀. Immunoblotting results indicated that the molecule had an electrophoretic mobility equal to that of intercellular cell adhesion molecule-1 (ICAM-1). Binding inhibition assays, however, showed that inhibition of binding of enriched CD34 cells by 3E₁₀was more prominent in comparison with that of ICAM-1.     

The p53 network: cellular and systemic DNA damage responses in aging and cancer

Genome instability contributes to cancer development and accelerates age-related pathologies as evidenced by a variety of congenital cancer susceptibility and progeroid syndromes that are caused by defects in genome maintenance mechanisms. DNA damage response (DDR) pathways that are mediated through the tumor suppressor p53 play an important role in the cell-intrinsic responses to genome instability, including a transient cell cycle arrest, senescence and apoptosis. Both senescence and apoptosis are powerful tumor-suppressive pathways preventing the uncontrolled proliferation of transformed cells. However, both pathways can potentially deplete stem and progenitor cell pools, thus promoting tissue degeneration and organ failure, which are both hallmarks of aging. p53 signaling is also involved in mediating non-cell-autonomous interactions with the innate immune system and in the systemic adjustments during the aging process. The network of p53 target genes thus functions as an important regulator of cancer prevention and aging.     

Midbrain dopamine neuron differentiation: factors and fates

Dopaminergic neurons in the midbrain (mDNs) play a central role in the regulation of voluntary movement as well as other complex behaviors, and their loss is associated with Parkinson's disease (PD). The development of functional mDNs from multipotent progenitors is orchestrated by cell-intrinsic factors and cell-extrinsic environmental cues in a series of stages: early midbrain patterning, specification of mitotic precursors, postmitotic mDN development, and functional maturation. Of particular interest is how extracellular information is integrated with cell-intrinsic developmental programs. Cell fate mapping studies suggest that the stem-like progenitors for mDNs reside at the ventral midline floor plate, a region that also serves as a source of inductive signals for mDN specification such as Sonic Hedgehog (SHH). Cell replacement therapies, and in particular the use of embryonic or adult stem cell-derived dopaminergic neurons, offer potential novel treatment venues for PD, but such strategies require a detailed understanding of mDN development.     

Wnt signaling and the regulation of stem cell function

Canonical Wnt signaling plays a crucial role in controlling cell expansion in many types of stem cells. Recent studies, however, demonstrated that Wnt is not only a general stem cell growth factor but can also influence cell lineage decisions in certain stem cell types by promoting specific fates at the expense of others. Thus, Wnt signaling elicits multiple functions in stem cells. Wnt activity appears to depend on cell-intrinsic properties that might change with time during development, thereby altering the cellular response to Wnt. Moreover, the spatial context of a stem cell also determines how the cell interprets Wnt signal activity, in that synergistic or antagonistic signaling pathways can modulate Wnt signaling. How a stem cell integrates Wnt and other signals and how such signaling networks regulate stem cell function on the molecular level remains to be elucidated.     

Innovative method for quantification of cell-cell adhesion in 96-well plates

Cell adhesion is an important part of many complex biological processes. It plays crucial roles in cancer, development and maintenance of stem cell compartment. The measurement of adhesion under experimental conditions might provide important information for cell biology. There are several protocols to measure adhesion, usually based on washing or shaking to remove non-adherent cells. Here, we describe a quantification method based on gravitational force to measure adhesion in a 96-well format. Non-adherent cells are separated and only vital cells are quantified with a colorimetric assay. This assay can be used especially when the “anti-adhesion” effect is present only for a short period of time like is the case of peptides or cytokines since it provides a trap for non-adherent cells in a way that they can not touch again the adherent surface. As examples we provide the quantification of cell-cell interaction with blocking antibodies anti-CD44 in hematopoietic stem cells and the effect of the stromal cell derived factor-1 (SDF-1) in the Jurkat cell line when they are in contact with mesenchymal stromal cells. This method facilitates fast and reliable measurement of cell adhesion in multiwell format for screening assays.Key words: adhesion assay, adhesion, SDF-1, CD44, hematopoietic stem cells, leukemia cell lines     

BMP signaling and stem cell regulation

Stem cells play an essential role in cellular specialization and pattern formation during embryogenesis and in tissue regeneration in adults. This is mainly due to a stem cell's ability to replenish itself (self-renewal) and, at the same time, produce differentiated progeny. Realization of these special stem cell features has changed the prospective of the field. However, regulation of stem cell self-renewal and maintenance of its potentiality require a complicated regulatory network of both extracellular cues and intrinsic programs. Understanding how signaling regulates stem cell behavior will shed light on the molecular mechanisms underlying stem cell self-renewal. In this review, we focus on comparing the progress of recent research regarding the roles of the BMP signaling pathway in different stem cell systems, including embryonic stem cells, germline stem cells, hematopoietic stem cells, and intestinal stem cells. We hope this comparison, together with a brief look at other signaling pathways, will bring a more balanced view of BMP signaling in regulation of stem cell properties, and further point to a general principle that self-renewal of stem cells may require a combination of maintenance of proliferation potential, inhibition of apoptosis, and blocking of differentiation.