Niche-Mediated Regulation of Stem Cell Functions
Fundamental stem cell processes, including self-renewal and differentiation trajectories, are regulated by the integration of chemical and physical signals present within local microenvironments, or niches (Figure 2). These signals are delivered in the form of soluble factors (e.g., growth factors, hormones, metabolites, oxygen), or as insoluble cues (e.g., cell-cell interactions, extracellular matrix, surface-bound signaling molecules), and often exhibit both spatial and temporal dynamics. In vivo, specialized niches direct resident adult stem cell function based on the combinatorial presentation of such microenvironmental stimuli. One of the most characterized adult stem cell niches is the HSC niche in the bone marrow. Numerous cellular components in the bone marrow, including osteoblasts, endothelial cells, and various mesenchymal cell populations have been demonstrated to influence HSC function.127 Substantial research efforts continue to be focused on resolving the cooperative influence of these diverse cell–cell interactions, and toward gaining an improved understanding of the role of HSC anatomical localization within distinct regions of the bone marrow compartment. Furthermore, cells differentiated from HSCs, such as macrophages, can provide feedback signals influencing HSC functions, consistent with the presence of important progeny–stem cell interactions in many stem cell niche contexts.128 In parallel to in vivo investigations of stem cell niche composition and dynamics, the development of engineered culture models (discussed in detail below), represents a promising strategy for elucidating key intercellular signals within stem cell microenvironments.
Figure 2. Engineering approaches for the study of stem cell microenvironments. Depicted are the prototypical components of a stem cell microenvironment, or niche. A network of interacting microenvironmental signals regulates stem cell functions. Bioengineering approaches, such as microtechnology tools, biomaterial fabrication, and computational methods coordinately enable the deconstruction of these complex microenvironments for systematic investigation, and the construction of engineered microenvironments for translational applications.
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Based on the role of the microenvironment in collectively shaping stem cell functions, the development of niche-targeted therapeutic interventions is considered an increasingly viable approach. For example, factors which influence bone marrow adipogenesis,129 or bone marrow osteoblast numbers,130,131 have been shown to affect hematopoiesis, and have been considered as therapeutic options for improving transplantation efficacy. Such strategies are analogous to microenvironment-directed treatments that have been developed for cancer therapy. In particular, several clinical treatments for breast cancer, including inhibitors of aromatase enzymes, angiogenesis inhibitors, and HER family receptor antagonists target stromal interactions.132 Further, the importance of microenvironmental context in cancer stem cell processes has been highlighted by studies analyzing the bidirectional interactions between cancer stem cells and both ‘normal’ and tumorigenic niche components. These interactions have been demonstrated to influence tumor growth and metastasis.133 In addition, the effect of microenvironmental perturbations on the self-renewal and directed differentiation of ES and iPS cells is a highly active field of investigation. An improved understanding of the extracellular signals regulating the differentiation of pluripotent stem cells and stem cell-derived progenitors can provide insights into normal developmental mechanisms and aid in optimizing differentiation protocols for cell sourcing applications. In particular, the application of engineering technologies can enable the deconstruction of the complex spatiotemporal mechanisms regulating stem cell fates (Figure 2), and establish the basis for the development of highly functional in vitro models of tissue development and homeostasis.
Engineering Approaches for the Controlled Presentation of Microenvironmental Signals
Engineered microsystems are designed to present microenvironmental stimuli in a tightly controlled manner, which facilitates the decoupling of signals for systematic examination. Such platforms have found great utility in the in vitro analysis of cell– cell and cell–ECM interactions involved in stem cell regulation. Specifically, in order to control cellular positioning, shape, and exposure to ECM proteins within two-dimensional cultures, a process termed micropatterning can be employed, which was developed based on tools used in the semiconductor industry, and can effectively pattern ECM proteins on a surface with micrometer scale resolution.134,135 This patterning is typically achieved with either photolithography methods, which are based on exposing a photosensitive material to UV irradiation through a patterned mask, or with soft lithography techniques, in which molecules are transferred to a surface using a patterned stamp made from silicone rubber, polydimethylsiloxane (PDMS). Numerous studies have employed micropatterned substrates to investigate the influence of cell shape and cell–ECM interactions on cellular functions due to the capability to independently manipulate the degree of cell spreading and cell–ECM contact.136 In a series of reports examining mesenchymal stem cell (MSC) differentiation, it was demonstrated that individual cell shape and spreading regulate cytoskeletal tension and associated Rho GTPase signaling, which influences the differentiation of these cells toward the osteogenic or adipogenic lineages.137–140 Furthermore, the impact of multicellular organization on determining osteogenic/adipogenic differentiation has been illustrated by studies utilizing ECM domain micropatterning to generate multicellular islands of predetermined geometries.141,142 These findings were similarly suggestive of the important role of cytoskeletal tension in this differentiation process. In addition to surface micropatterning, PDMS stamps can be utilized for the microscale molding of hydrogels such as agarose, polyacrylamide, and poly(ethylene glycol) (PEG) into diverse geometries including microwell structures. Microwells have been widely used for high-throughput analysis (discussed in detail below) and as a tool to investigate cell–cell interactions. For example, a platform incorporating bowtie shaped agarose microwells was developed to examine the effects of homotypic interactions between two adjacent cells, while decoupling the influence of cell-cell contact from the effects of cell spreading.143,144 In order to examine the role of cell–cell interactions in ES cell differentiation, both microwell and micropatterning platforms have been utilized for the formation of ES cell aggregates with tightly controlled diameters. These systems, as well as complementary suspension culture techniques, have collectively demonstrated the role of aggregate size in directing the early lineage commitment of ES cells.145–153
The mechanical properties of the stem cell microenvironment can also significantly influence stem cell function. For instance, various mechanical perturbation regimens have demonstrated the effects of mechanical strain on the self-renewal of human ES cells,154,155 the vascular differentiation of mouse ES cells,156 and the myogenic differentiation of MSCs.157 Furthermore, MSC fate decisions as well as the self-renewal capabilities of skeletal muscle stem cells have been demonstrated to be regulated by substrate rigidity, which is sensed as a mechanical signal.158,159 In order to systematically explore the effects of substrate elasticity characteristics on cellular functions, hydrogel materials including polyacrylamide,160,161 PEG,159 PDMS,162 and hyaluronic acid (HA)163 are commonly utilized as culture surfaces that exhibit tunable elastic moduli upon alterations in the degree of crosslinking. Although polyacrylamide and PEG hydrogels are hydrophilic and generally resist protein adsorption, they can be chemically modified to present either native ECM proteins or adhesive peptides for mediating cell adhesion. In addition to bulk modifications, microtechnology methods have been applied toward the fabrication of micropillar systems which enable the modulation of substrate rigidity without altering the inherent material properties.164–168 In this approach, PDMS is molded into an array of vertical posts on top of which cells can be cultured. The substrate rigidity is then defined by the post dimensions, which are modular and can be adapted for the desired elastic characteristics. Analysis of MSC differentiation on a micropillar array164 demonstrated effects of substrate rigidity on lineage commitment, consistent with polyacrylamide studies,158 and facilitated correlations between traction force (calculated by the deflection of the micropillars), focal adhesions, and cytoskeletal tension.
Substantial efforts have also focused on the development of improved three-dimensional (3D) culture platforms. Highly functional 3D model systems can provide the capability to investigate cellular processes which can be affected by dimensional context, such as growth factor and adhesion receptor signaling, and other complexities in cellular organization and mechanics which are present in 3D tissue architectures.169–173 Many natural/biologically derived and synthetic biomaterials have been extensively examined toward the development of 3D scaffolds that recapitulate the ECM of a relevant tissue or provide specific empirically determined characteristics. For example, a photopolymerizable hyaluronic acid hydrogel platform has been demonstrated to support the self-renewal of human ES cells in 3D, without a requirement for feeder cells.174 In addition, in order to direct or augment the differentiation of both pluripotent and various adult stem cell populations toward specific lineages in a 3D context, a broad range of scaffold materials have been explored, and findings from these studies are summarized in several comprehensive reviews.175–177 Notably, recent advances in biomaterials have significantly improved the capability to present a diverse range of biological signals to cells in 3D. In particular, synthetic hydrogels, such as PEG-based systems, are highly tunable in regards to porosity and mechanical properties, and can be co-encapsulated or conjugated with bioactive factors to add biological functionality in a tightly controlled manner. For instance, the integration of small functional groups has been shown to influence cell-secreted growth factor and ECM presentation,178–181 and various peptide sequences can be incorporated to dictate cellular adhesion and cell-mediated degradation of the hydrogel.182 Releasable factors can also be introduced by the co-encapsulation of microparticles within the 3D network.183–185 Further, novel strategies for the dynamic modulation of hydrogel factor presentation, based on photoactuation chemistries, have been recently demonstrated.186–190 In one study, the temporal control of RGD peptide presentation through a photocleavage process facilitated the chondrogenic differentiation of encapsulated MSCs.190 Collectively, such developments continue to enhance the overall flexibility in the design of scaffold systems, and significantly increase the number of parameters which can be manipulated to influence stem cell function in 3D environments.
High-Throughput Analysis of Stem Cell Microenvironments
As highlighted in previous sections, robust high-throughput analysis methods are central to the elucidation of stem cell genetic regulation at the genomic scale. In an analogous manner, microtechnology approaches have been applied to the development of high-throughput platforms that enable the systematic screening of microenvironmental signals. The fabrication of cellular microarrays represents an example of one of these approaches aimed at exploring, in parallel, a range of combinations of signals that cannot be practically evaluated with standard techniques. Cellular microarrays typically consist of either printed spots of biomolecules, including adhesive components and other signaling/detection elements, onto which cells are seeded, or alternatively, consist of directly printed arrays of live cells encapsulated in hydrogel droplets. Currently, cellular microarrays have been most broadly applied to the assessment of ECM effects, and in particular, the role of combinations of ECM proteins in mediating cell processes.191–196 Together with ECM molecules, microarrays of cell surface ligands and growth factors have been developed, and have provided intriguing insights into how neural197 and mammary progenitor198 differentiation is regulated by combinatorial signals. In addition to natural proteins, microarrays of biomaterials have been utilized to identify synthetic surfaces, with distinct polymer chemistries, which can significantly influence pluripotent and adult stem cell self-renewal and differentiation.178,199–203
Microwell platforms have also been broadly used for the high-throughput analysis of stem cells.204 Microwell arrays are typically fabricated through direct etching of hard materials (e.g. glass, silicon) or through a combination of photopolymerization and soft-lithography-based molding of hydrogels. In particular, this approach has been applied to the analysis of individual stem cells in order to evaluate clonal heterogeneity.159,205 Hydrogel microwells can be functionalized with biomolecules, as highlighted by a recent strategy that paired microwell molding with protein microarraying to analyze neural stem cells and MSCs within microwell arrays presenting a range of combinatorial stimuli.206 Microfluidic-based approaches, integrating microwells or hydrodynamic traps, have also been employed to generate cellular arrays.207–210 In addition, the emerging field of droplet microfluidics211 represents an attractive approach to increase the throughput of 3D fabrication, while maintaining precise control of the environmental components. For example, miniaturized cell-encapsulated hydrogels were fabricated with microfluidic methods in order to examine co-cultures,212 incrementally modulate material stiffness,213 and generate microscale constructs for the assembly of larger patterned structures.214 Furthermore, a recent study described a high-throughput 3D analysis approach, analogous to flow cytometric measurements of individual cells, which could enable the sorting of miniaturized stem cell constructs based on the expression of a differentiation reporter, and was additionally compatible with multiplexing and in vivo assessment.215
Engineered Environments for Optimized Expansion and Differentiation
Building on the mechanistic studies and the increasing capability to precisely control in vitro culture environments, significant research efforts are focused on the development of platforms which could promote the proliferation and directed differentiation of stem and progenitor cells for clinical applications.216 Notably, microfluidic platforms have provided key insights into the influence of flow and nutrient transport on stem cell processes. For example, microfluidic devices, which exhibit spatiotemporal control of perfusion characteristics, were utilized to investigate the effects of hydrodynamic shear and nutrient delivery on ES cell proliferation and differentiation.217–219 Consistent with these microfluidic studies, larger scale perfusion systems have demonstrated that nutrient transport and the retention of autocrine soluble factors, which are collectively determined by the flow characteristics, can significantly influence ES cell proliferation.220,221 Moving forward, a significant challenge in the field is the development of approaches that can adequately translate the combinatorial control of microenvironmental signals achieved at the microscale in laboratory models, to a scale that can attain clinically effective regulation of stem cell proliferation and directed differentiation.
Computational approaches can be employed to assist in the interpretation of combinatorial perturbations. In particular, advanced statistical methods and network models have been applied to pairwise and higher order combinations of signals to evaluate and predict synergistic effects.222–224 Such tools can greatly complement engineered high-throughput platforms for the deconstruction of complex environments. Computational tools, such as learning algorithms, have also been shown to aid in the refinement of experimental conditions, including the iterative identification of a distinct combination of small molecules which support human ES cell self-renewal.225 In addition, toward a more complete understanding of microenvironmental regulation and the optimization of in vitro stem cell platforms, computational models of intercellular communication networks can be formulated. For example, as mentioned previously, HSC expansion has been shown to be regulated by feedback signals from differentiated HSC progeny, and systems models of intercellular interactions have been developed for this process.226,227 By further incorporating models of bioreactor conditions, a recent study computationally predicted parameters that could enhance HSC proliferation, which were confirmed through in vitro expansion experiments.126 Further, intercellular signaling models can be formulated for in vivo developmental processes, such as the recent demonstration of a Wnt-BMP signaling circuit in tooth organogenesis,228 which was predicted based on the integrated analysis of epithelial and adjacent mesenchymal cell gene expression profiles, and validated with genetic mutations in a mouse model. In the future, it is anticipated that systems-based analysis of the multilineage differentiation and patterning of stem cells within in vitro culture models will provide key insights into developmental mechanisms and relevant abnormalities.