Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)
Part I. Stem Cells
Various populations of stem cells reside in the body, undergoing continuous self-renewal throughout the organism’s lifetime. The complex milieu comprising many types of cells and the ECM, as well as the signalling molecules associated with each population of stem cells, is collectively termed the ‘stem cell niche’. Since the stem cell niche controls the fate of stem cells in different tissues, its cellular and molecular organization is very important.
The cellular composition of supportive stromal cells varies in different types of stem cell niches. The HSC niche contains different cell types, including osteoblasts, osteoclasts, reticular cells, vascular cells, MSCs and neuron-Schwann cells (Adams and Scadden, 2006; Wang and Wagers, 2011). The intestinal stem cell (ISC) niche is less complex, in that the epithelial monolayer comprises one type of absorptive cell (enterocyte) and four types of secretory cells (Goblet, Paneth, enteroendocrine and Tuft cells). The mucosal surface forms crypts, which project downward, and villi, which project upward. Stem cells have been identified in the crypt of the small intestine, which consists of the duodenum, jejunum and ileum (Ema and Suda, 2012).
Figure 3.1. The stem cell niche is a specialized and dynamic microenvironment in which a number of inputs regulate stem cell behaviour. This schematic illustration summarizes the components of a typical stem cell niche: the stem cell itself, stromal supportive cells, soluble factors, ECM, vascular network and cell adhesion components (cell – cell and cell – ECM). Although many stem cell niche constituents are conserved in tissues, it is unlikely that every stem cell niche in every tissue includes all of the components listed. Different niches, for different types of stem cells, can combine a selection of these components for proper self-renewal and differentiation.
The niche for muscle stem cells, comprising satellite cells, is simpler. Satellite cells (about 5% of adult muscle nuclei) are quiescent muscle precursors, forming a small population beneath the basal lamina of each myofibre (Mauro, 1961). The satellite cell niche contains a basement membrane and muscle fibres and/or endothelial cells in close proximity, which can function as supporting stromal cells (Dhawan and Rando, 2005; Christov et al., 2007).
ECM components make up the constitutive part of the niche. The ECM is a complex assembly of many proteins and polysaccharides, forming an elaborate meshwork in a three-dimensional organization within tissues (Hay, 1991). Accordingly, the diversity of ECM components includes both large secreted glycoproteins and small secreted protein factors, either diffusing or associated with the ECM or cell membrane. In general, ECM proteins are highly asymmetric in shape, and when they are combined, a functional network results (Hay, 1991; Mecham, 2011).
The ECM comprises several secreted proteins, including collagens, fibronectin, elastin and fibrillins. Although the ECM is formed by a large variety of proteins and polysaccharides with different structures and functions, some common features are evident. Many ECM proteins are very large, their size often contributing to extensive glycosylation. In particular, proteoglycans (PGs) contain long, charged glycosaminoglycan (GAG) chains covalently attached to serines or threonines of the core protein. Some GAG chains are also found unconnected to a protein, such as hyaluronan, a major nonproteinaceous GAG component of ECM. Hydration of these carbohydraterich components exerts a swelling pressure against the surrounding fibrous network, providing tissue turgidity and compressibility and facilitating molecular transport (Hubmacher and Apte, 2013).
All ECM molecules are multidomain elements, in which different or equal domains are arranged in a specific domain organization. Domains are defined as homologous units, the homology following from sugar or amino acid sequence comparisons. Even homologous domains may have large sequential and structural differences, which present rather different functions. The combination of different domains leads virtually all ECM components being multifunctional. Commonly, several GAG and protein domains act in a coordinated fashion. Domains also interact such that the resultant multidomain elements are able to form new functional entities. This multifunctionality and the resultant expanded shapes provide the potential for lateral interactions, favouring the formation of fibres and other supramolecular assemblies of ECM components (Mecham, 2011).
Growth factors are key components of the stem cell niche. It is well known that secreted growth factors play a key role in coordinating many biological functions, such as stem cell growth, division, differentiation, apoptosis and signalling. Some signals mediate communication between direct neighbours (juxtacrine) or over several cell diameters (paracrine), whereas others act on distant tissues or organs (endocrine). Many individual growth factors are themselves pleiotropic, exerting multiple actions in different cell types, and redundant where many different growth factors have overlapping actions. Growth factor pleiotropy and redundancy can be respectively explained, at least partially, by the ability to signal via more than one type of receptor complex and by the sharing of an individual receptor component (Ozaki and Leonard, 2002; Sizonenko et al., 2007).
Studies in flies indicate that supportive stromal cells secrete growth factors, such as paracrine signals, that are required for maintenance of stem cell identity and for specification of stem cell self-renewal (Xie and Spradling, 2000; Gonzalez-Reyes, 2003; Losick et al., 2011). Extracellular signals, such as Notch, Wnt and Sonic hedgehog (Shh), have been associated with self-renewal and maintenance of HSCs (Maillard et al., 2003; Reya and Clevers, 2005). However, rather than being expressed by surrounding stromal cells in bone marrow (paracrine signalling), Wnt may be secreted from the HSCs themselves and thus might act through autocrine signalling to control stem cell fate (Van Den Berg et al., 1998; Malhotra and Kincade, 2009).
Extracellular growth factors signal from both soluble growth factors and ECM-bound factors. The ECM contains growth factor-binding GAGs, heparan sulfate (HS) and chondroitin sulfate (CS). Perlecan is a ubiquitous heparan sulfate proteoglycan (HSPG) found in basement membranes and cartilage that binds multiple regulatory factors, including fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and interleukin 2 (IL-2), to concentrate them and form morphogen gradients. Perlecan knockout mice have revealed that cartilage perlecan is essential for vascularization of the perichondrium. Cartilage perlecan promotes the activation of VEGF/vascular endothelial growth factor receptor (VEGFR) by binding to the VEGFR of endothelial cells (Yayon et al., 1991; Ishijima et al., 2012), providing gradients of soluble factors that influence cell growth and the differentiation, motility and viability of stem cells.
Syndecans are type I transmembrane cell-surface HSPGs. Four syndecan family members occur in vertebrates: syndecan-1, -2, -3 and -4. Syndecans are expressed at up to 1 million copies per cell in every nucleated cell of the organism. Most cell types, with the exception of erythrocytes, express at least one syndecan family member, and a few can express all four. Syndecan-1 is expressed in epithelial cells and plasma cells; syndecan-2 is mainly expressed in fibroblasts, endothelial cells, neurons and smooth-muscle cells; syndecan-3 is primarily found in neuronal tissues, but it is also important for chondrocyte proliferation; while syndecan-4 is nearly ubiquitous (Woods et al., 1998; Couchman, 2003; Choi et al., 2011). Syndecans are expressed by stem cells and their supportive stroma, as well as cancer cells (Chen et al., 2004; Drzeniek et al., 1997). The protein cores of syndecans consist of a highly conserved C-terminal cytoplasmic domain, a single-pass transmembrane domain and a large N-terminal extracellular domain (Bernfield et al., 1999; Couchman, 2010). GAG chains are present on syndecan ectodomains, which carry up to five GAG chains. Syndecan-1s from different tissues display different GAG types, comprising HS and CS of varying lengths and fine structures (Sanderson and Bernfield, 1988; Bernfield et al., 1992).
Cell-surface HSPGs bind extracellular proteins and form signalling complexes with receptors for growth factors (Yayon et al., 1991; Bernfield et al., 1999). This ligand binding is a means of regulating the occupancy and response of the specific receptor (Harmer, 2006). Outcomes seem to depend on whether the ligand is soluble (i.e. a growth factor) or insoluble (i.e. a cell or ECM component), whether the ligand also interacts with a signalling receptor and whether the ligand binds to the HS chains on HSPGs or their core proteins. HSPG ectodomains are shed from the cell surface, resulting in soluble PGs that, presumably, retain all the binding properties of their parent molecules. Shedding can rapidly reduce HSPGs at the cell surface, thereby enabling the shed ectodomains to compete for ligands with their cell-surface counterparts. Therefore, shedding provides a means of regulating all HSPG – ligand interactions. Because the soluble ectodomains can interact with any extracellular molecule, they can have activities other than those shown by the cell-surface PGs (Gallagher, 2001; Harmer, 2006). It has been proposed that HS binds soluble protein ligands from three-dimensional space, converting them into a two-dimensional array that enhances further molecular encounters (Lander, 1998). These proteins might also be protected from degradation by binding to GAG, preventing oligomerization and/or conformational changes that facilitate further interactions. One example is the interaction between FGF2 and HS that is required for binding of FGF2 to its high-affinity tyrosine-kinase receptor (Yayon et al., 1991). A ternary complex of growth factor, HS and cell-surface receptor seems to maximize signalling potential (Couchman, 2003). HSPGs are considered the most common and widely acting low-affinity receptors on the cell surface, and they play a central role in the reception and modulation of a wide range of growth factors (Tamm et al., 2012).
Growth factors also bind to specific domains of ECM proteins. For example, fibronectin and vitronectin bind to hepatocyte growth factor (HGF) and modulate its biological activity (Rahman et al., 2005). Fibronectin also binds to VEGF, enhancing specific cellular responses to it (Wijelath et al., 2006). Finally, fibronectin has been found to bind platelet-derived growth factor-BB (PDGF-BB), a key factor for the survival of mesenchymal cells (Lin et al., 2011). Type IV collagens bind to bone morphogenetic protein (BMP) and regulate its activity, enhancing signalling in target cells (Wang et al., 2008). The PG agrin binds BMP2, BMP4 and transforming growth factor-β1 (TGFβ1) with relatively high affinity. This inhibits the activity of BMP2 and BMP4, but enhances the activity of TGFβ1 (Banyai et al., 2010).
The biological activity of many growth factors can be intrinsically related to their interaction with ECM elements of the niche. The degree of regulation of growth factor activity is dependent on adequate levels of ECM components, which can vary in different tissues. Such activity can promote niche specificity in certain tissues, such as bone marrow and brain, as well as support specific SC differentiations, including, for example, into HSCs and neuronal stem cells.
Two essential mechanisms are closely related during embryogenesis: patterning and growth. Since the ECM can act as a reservoir of growth factors (e.g. VEGF, Wnt, BMP and FGF), such factors form gradients that control pattern formation during developmental processes. ECM binding of these factors can influence these gradients. Morphogens (secreted signalling molecules, including growth factors, that regulate the size, shape and patterning of animal tissues and organs) are conserved molecules. It seems reasonable that morphogen gradients incorporate ECM binding as part of their regulation (Wartlick et al., 2009; Yan and Lin, 2009). Morphogen binding to molecules in the extracellular space affects signal movement. The diffusion of a particle that is interacting with binding partners in this manner is referred to as ‘effective diffusion’ (Crank, 1979). Interactions with binding partners can modify ligand dispersal and activity in at least four ways:
(i) altering the mobility/diffusivity of a signal, (ii) concentrating ligand at the surface of a cell, (iii) promoting or hindering ligand – receptor interactions, and (iv) influencing the extracellular stability of a ligand (Mьller and Schier, 2011). Moreover, the niche can generate high or low local gradients of growth factors, in which the ECM molecules elicit specific biological responses by the juxtaposition of growth factors and their cell-surface receptors in a confined spatial environment, resulting in short-range paracrine or autocrine growth factor signalling. This can lead to different stem cell responses, depending on the local level of growth factor.