Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research

Abstract

Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity), viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D) and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement), and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems.

Figure 1

Physical, spatial, and molecular aspects of extracellular matrix that are known to affect stem cell behavioral patterns and choices. Extracellular matrix (ECM) of mammalian tissues in vivo is a complex structure composed of multiple molecular components, such as fibrils, fibril-associated crosslinking elements, and specific ligands interacting with cell receptors. Such molecular complexity has a biological reason, since lack of ECM molecules, due to mutation or knockout, often results in pathology or even mortality. Molecular composition of a matrix composition and the way of structural arrangement of the molecular components determine the physical, spatial, and molecular characteristics of the scaffold which, as we demonstrate in the review, may actively affect stem cells behavioral patterns. Physical aspects include stiffness (or elasticity); viscoelasticity; pore size and porosity; amplitude of static and dynamic deformations of the matrix (tensile, compressive, or shear); and frequency of cyclic deformations. Due to complex organization, elastic properties of the natural ECM cannot be characterized by a single parameter of Young's modulus (which is valid for many synthetic gels). The stress-strain relation is often nonlinear and is described by stress-strain curve; the natural ECM tend to rearrange their structure under stress, which makes them viscoelastic and prone to plastic deformation. Viscoelastic materials change their elastic properties when they are subject to static strains or cyclic (dynamic) deformations; therefore, one has to take tensile characteristics of the system into account. Spatial arrangement includes dimensionality (2D or 3D) of the scaffold introduced to the cell; thickness of the substrate layer underlying the cell; cell polarity; surface area and geometry of adhesion surface; microscale topography of the surface; epitope concentration; epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and order or disorder in epitope arrangement); size, shape, and level of disorder of nanotopographical features such as fibers diameter and orientation. Molecular properties concern structural complexity of ECM molecules, types of adhesion epitopes and corresponding receptors, co-signaling (cooperation of growth factor- and matrix-dependent receptors), and affinity interactions.

Figure 2

Interactions of the cell structures with adhesive matrix epitopes: from cell-size scale to nanoscale. (a) Certain events of matrix perception by the cell occur on scale comparable with size of the cell. Cells distinguish between 2D and 3D presentations of the epitopes. In case when 3D matrix is presented only from the basal side of the cell, thickness of the matrix layer can be critical for cell perception of the matrix. Cell perception of the underlying matrix thickness ranges approximately from 5 to 20 micrometers, which is comparable with average cell diameter. In cases when matrix defines the geometrical properties of cell adhesion surface, such as in case of adhesion islands model, adhesion area and shape can be influential factors for stem cell behavioral choices. (b) Cell propagates and spreads on the matrix due to spreading of filopodia, which form stable adhesion contacts with the matrix. In order to achieve stability of such adhesion contact, it takes not a single epitope but cluster of epitopes which, in turn, enable clustering of cell receptors and signaling to the cell. The following parameters of epitope clusters may be important to influence cell behavioral choices: number of epitopes per cluster, space between clusters, and so forth. Topography on micro- and nanoscale can also be influential on cell behavior. (c) Such events like co-signaling (synergy between the different cell receptors, i.e., receptor for matrix epitopes and receptor for growth factors) occur on even smaller scale. Importantly, the natural matrix adhesion molecules are very large ones (molecular weight up to 1 million Daltons, length up to 300 nanometers), compared to typical adhesion peptides (composed of several amino acids, with molecular weight below 1 thousand Daltons). Apparently, one has to consider geometry of functional clusters of receptors and matrix molecules on molecular level. (d) Affinity and specificity of matrix adhesion epitopes interactions with cell receptors occur on even smaller scale.

Figure 3

Laminins: a model system to investigate roles of molecular complexity of natural matrix molecules. Natural extracellular matrix molecules are very large and complex molecules, often composed of more than one polypeptide chain (collagens have 3 chains, laminins have 3 chains, and fibronectin has 2 chains) and multiple domains with distinct adhesive and geometrical properties. Mutations in either chains or domains often result in severe pathology. Apparently, such molecular complexity developed during evolution for a reason, and natural ECM molecules have more function, compared to small adhesive peptides. However, it is not easy to establish roles for each specific domain and structure of the natural large ECM molecules. Family of laminins, due to their molecular versatility, is a perfect model system to investigate reasons for functional complexity of matrix molecules. Laminins are the natural adhesion ligands for many stem cell types, like embryonic stem cells (ESC), hematopoietic stem cells, sperm stem cells, and probably many others. Due to unique composition of three heterogeneous chains, each of which can be varied, cell adhesion domains undergoing natural proteolytic maturation and, therefore, change in affinity and specificity, availability of conditional knockout models, and mutated proteins in laminin family are an excellent system to investigate reasons for molecular complexity of natural matrix molecules. Laminins are large, heterotrimeric molecules that comprise one α, one β, and one γ chain. Size of laminin trimer varies from 400 to 1000 kDa. Five α (α1–α5), four β (β1–β4), and three γ (γ1–γ3) chains are known in mammals. Laminin-521 (LM-521) consists of α5, β2, and γ1 chains. The molecule in the figure represents the cross-shaped laminin isoform; however, some laminin isoforms have truncated shapes: Y-like or rod-like shape. The α1, α2, α3B, and α5 trimers are cross-shaped, while the α3A and α4 trimers are Y-shaped or rod-shaped. Short arms of laminins (N-terminal parts of α, β, and γ chains) can bind other laminins short arms and other ECM proteins. Laminins in solution are capable of self-assembly via N-terminal short arms in the presence of calcium.

Figure 4

Biological relevance as a key to developing functional in vitro culture systems. There is a need for developing advanced, highly functional, robust, and long-term lasting in vitro culture systems for cells and organoids. In order to identify biologically active scaffolds, adhesion epitopes, and growth factors, high-throughput array approach is often used. It allows unbiased screening of large libraries of soluble compounds (proteins, peptides, and inorganic substances), as well as libraries of matrix scaffolds. However, sometimes biologically active compounds show false negative result, if the other aspects of the system are not biologically relevant. (a) The natural niche of a specific cell type can often serve a prototype for developing a highly functional in vitro culture system. Synergy of biologically relevant extracellular matrix cues and growth factors may be required in order to enable well-regulated cell function. (b) When a library of growth factors is analyzed in cell-based high-throughput screening array, the highly biologically active compounds may be identified as “false negatives” if the adhesive scaffold is not biologically relevant and does not enable co-signaling. (c) Also, if a library of scaffolds, whether natural, artificial, or mixed, is analyzed in cell-based high-throughput screening array, the truly functional scaffold may fail to provide the desired effect, if the cell culture medium lacks biologically relevant soluble factors that take part in co-signaling. (d) It is advisable, therefore, to arrange the high-throughput screening assays that would screen for a combination of growth factors library versus a scaffold library. The positive hit that can be missed in single-library screening may be identified in double-library cross-screening array.