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Ann Biomed Eng. Author manuscript; available in PMC 2012 June 1.
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PMCID: PMC3365657
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Engineering Approaches Toward Deconstructing and Controlling the Stem Cell Environment
Faramarz Edalat,1,2 Hojae Bae,1,2 Sam Manoucheri,1,2 Jae Min Cha,1,2 and Ali Khademhosseini1,2,3
1Center for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, Massachusetts 02139, USA
2Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
3Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts 02115, USA
Correspondence: Ali Khademhosseini, Partners Research Building, Room 252, 65 Landsdowne Street, Cambridge, Massachusetts 02139, USA. alik/at/rics.bwh.harvard.edu
Small right arrow pointing to: The publisher's final edited version of this article is available at Ann Biomed Eng
Abstract
Stem cell-based therapeutics have become a vital component in tissue engineering and regenerative medicine. The microenvironment within which stem cells reside, i.e. the niche, plays a crucial role in regulating stem cell self-renewal and differentiation. However, current biological techniques lack the means to recapitulate the complexity of this microenvironment. Nano- and microengineered materials offer innovative methods to: (1) deconstruct the stem cell niche to understand the effects of individual elements; (2) construct complex tissue-like structures resembling the niche to better predict and control cellular processes; and (3) transplant stem cells or activate endogenous stem cell populations for regeneration of aged or diseased tissues. Here, we highlight some of the latest advances in this field and discuss future applications and directions of the use of nano- and microtechnologies for stem cell engineering.
Keywords: biomaterials, nano- and microfabrication, high-throughput, microfluidics, regenerative medicine
What distinguishes stem cells from other cell types is the capability to self-renew and differentiate into lineage-specific progenies. These characteristics have made stem cells a promising tool in the fields of regenerative medicine99 and cancer biology.18 However, despite their potential, the translation from laboratory to clinic has been slow.22 One reason is the inability to expand adult stem cells in vitro while preserving their differentiation capacity, and another is the lack of control over the differentiation of stem cells into desired cell types. To overcome these bottlenecks, it is crucial to understand the biology of stem cells and the molecular mechanisms governing stem cell self-renewal and lineage commitment.
Common stem cell types include embryonic,128 adult, and induced-pluripotent stem cells (iPSCs).125 Adult stem cells have a limited differentiation capacity (multipotent), meaning they are able to form several lineages within a tissue and are organ specific. For instance, the bone marrow houses mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), whereas neural stem cells (NSCs) reside in the subventricular zone and hippocampus.89 In contrast, embryonic stem cells (ESCs) and iPSCs are capable of producing all the cell types of an organism (pluripotent). ESCs are derived from the inner cell mass of the blastocyst of an embryo and iPSCs are generated by the genetic reprogramming of somatic cells into pluripotent stem cells.
It is becoming increasingly appreciated that the stem cell microenvironment, or niche, is responsible for regulating stem cell behavior and homeostasis.43, 94, 95, 117, 132 Indeed, in their niche, stem cells are maintained or can undergo proliferation and differentiation in response to injury, disease, or aging to replenish lost cells or tissue. This homeostatic function is governed by intrinsic (genetic and epigenetic) as well as extrinsic (environmental) biological stimuli. The discovery of the niche and the continual uncovering of its constituents have allowed scientists to study the function of each component by deconstructing the niche into its individual parts.11, 133 Recently, bioengineering methods have been instrumental in tackling biological questions that cannot be answered by conventional cell culture techniques.20, 121 In this regard, engineering principles drawn from materials science to microfabrication have emerged to be useful, not only in the simplification but also the construction of an in vitro stem cell niche.65
The niche is composed of several constituents that work together to modulate stem cell function (Fig. 1). Inside this microenvironment, stem cells are exposed to a milieu of extracellular matrix (ECM), support or hub cells, and soluble factors. ECM is made up of proteins and polysaccharides that form a cross-linked network and impart structural and mechanical integrity to tissues. However, their role extends beyond acting as scaffolds to providing ligands that interact with cell receptors, such as integrins, to mediate cell adhesion, shape, migration, apoptosis, self-renewal, and differentiation.80 Similarly, support cells interact with stem cells via membrane proteins. Soluble factors, such as cytokines, are another element that control stem cell behavior. Particular examples of such cytokines include wingless-related (Wnts)109 and hedgehog proteins,9 fibroblast growth factors (FGFs),25 and bone morphogenetic proteins (BMPs).143 Metabolic products, such as calcium, are another class of biological cues that affect stem cells. The impact of these biophysico-chemical components on the stem cell phenotype are an important design consideration in engineering the stem cell microenvironment, in vitro.
Figure 1
Figure 1
The stem cell microenvironment, or niche. In the niche, stem cells are exposed to ECM, soluble and immobilized molecules (cytokines, metabolic products), support cells, and physical cues (topography, structure, stiffness, static and dynamic forces). Stem (more ...)
In this review article, recent progress in stem cell engineering is highlighted. Specifically, we dissect the stem cell niche into its individual elements including scaffold, biophysical and biochemical factors, and describe how engineering approaches are being applied towards studying these elements. Furthermore, recent technologies that have been used or are promising for the fabrication of complex, tissue-like structures using stem cells are described. We conclude by introducing the role of advanced biomaterials in stem cell-based in vivo therapeutics.
The traditional 2-dimensional (2D) culture system has afforded experimenters with simplicity in the analysis of individual variables affecting cells. However, this simplification has led to a disjoint in the translation of in vitro results to in vivo systems. A 3-dimensional (3D) platform, while more complex, better mimics the in vivo organization of cells and is a crucial requirement for tissue construction.11, 105 The first step in this process is the selection of the appropriate material as the cellular scaffold. Currently, in vitro cell cultures often utilize rigid polystyrene surfaces or extracted ECM proteins, such as collagen or laminin. Also, the use of animal-based feeder layers is common in ESC cultures. These culture techniques lack 3D spatial complexity, are not amenable to chemical or physical modification, and employ xenogeneic cells. To overcome these impediments, advanced materials can be designed to incorporate cells and present biochemical signals, such as adhesive ligands and cytokines. Furthermore, mechanical properties, such as porosity, stiffness, and degradation are tunable. For detailed review of biomaterials in stem cell biology and tissue engineering, we refer the readers to several excellent reviews.24, 29, 82, 86, 115 As more discoveries are made regarding the role of biophysical and biochemical factors in stem cell regulation, the design of materials that incorporate these findings will enable more control over the fate of these cells.
Biomaterial-based scaffolds serve as a framework for a 3D stem cell niche in vitro. These materials will function as platforms for cell attachment, migration, proliferation and differentiation. In designing these materials, criteria that must be considered include biocompatibility, fluid transport for the exchange of gases and nutrients, and biochemical and mechanical integrity for cellular processes.
Biomaterials can have a variety of structures depending on their composition and processing methods. Hydrogels are one type of structure that can be fabricated, and this type of biomaterials has wide utility in stem cell biology. Hydrogels are a 3D network of cross-linked hydrophilic polymers (natural or synthetic) containing 95–99% water, whose physico-chemical properties are highly tunable.135 This class of materials is a particularly attractive cellular scaffold substitute given its inherent structural similarity to ECM.45, 122 Hydrogels are synthesized by the presence of a cross-linking agent through numerous schemes, such as free radical polymerization,134 Michael addition,83 or click chemistry.85 Microfabrication techniques have been utilized to cultivate hydrogels with desired microarchitectures. These techniques include emulsification,96 micromolding,140 photocrosslinking,53, 70 microfluidics,10, 101 and bioprinting.91 Besides hydrogels, other structures include fibrous6 or macroporous68 scaffolds, often formed through electrospinning or porogen leaching, respectively (Fig. 2a).
Figure 2
Figure 2
Engineered scaffolds for stem cell culture. (a) Scaffolds can be designed to have hydrogel,77 (Adapted with permission from Springer Science) fibrous,87 (Adapted with permission from American Chemical Society)or macroporous118 (Copyright (2000) National (more ...)
Nature-Derived Biomaterials
The material used for 3D stem cell culture can be naturally-derived or synthetic. Natural biomaterials are often acquired from isolating ECM components, such as collagen, hyaluronic acid (HA), and fibronectin, but are also derived from non-ECM materials, such as chitosan and silk.24 Gerecht et al.47 used chemically modified HA to form photopolymerizable hydrogels for human ESC encapsulation. Human ESCs encapsulated in these hydrogels maintained their pluripotency, and upon exposure to vascular endothelial growth factor (VEGF) differentiated to the vascular lineage. Recently, decellularized organs have also been used as scaffolds to generate tissue-engineered lung,106 liver,129 heart,100 and bone.50 For instance, bone grafts in the shape of the temporomandibular joint condylar bone were engineered using a bioreactor and decellularized trabecular bone seeded with human MSCs (Fig. 2b).50 The advantages of natural scaffolds are their biocompatibility and provision of biological cues, however batch-to-batch variability, potential adverse immunogenicity and poor control over physico-chemical properties limits the use of these materials.
Synthetic Biomaterials
Synthetic materials have come to fill the gap created by their natural counterparts due to greater control over their mechanical and chemical properties. The most widely used synthetic materials in stem cell engineering are polymers, particularly polyacrylamides, polyacrylates, polyethers and polyesters.86, 115 These scaffolds can be modified to include adhesion molecules, such as arginine-glycine-aspartate (RGD), which is a ubiquitous, integrin-binding domain of fibronectin58; additionally, growth factors36 and protease-sensitive peptide sequences for scaffold degradation can be incorporated into the scaffold (Fig. 2c).104 In designing these scaffolds, biodegradability is an important criteria under consideration; the rate of degradation ideally should be comparable to the rate of ECM deposition and tissue formation by the cells seeded within the scaffold. Polyesters, such as poly (glycolic acid) and poly(L-lactic acid) (PLLA), have long been used in biological settings, given their broad range of degradability, and that their degradation byproducts can be cleared through endogenous metabolic pathways.24 For instance, poly(lactic-co-glycolic acid)/PLLA copolymeric scaffolds have been used for human ESC differentiation and formation of structures resembling neural, cartilage, or liver tissues in the presence of various growth factors.76 In contrast to polyesters, poly(ethylene glycol) (PEG) does not biodegrade; one way of making this class of synthetic material biodegradable is through using a peptide cross-linker sensitive to proteases during the polymerization of PEG units.84 Degradation occurs as cells in the scaffold secrete proteases, such as matrix metalloproteases, cleaving the peptide cross-linkers. In addition, given the inert nature of PEG, it can be used as a base scaffold to present small organic molecules, peptides and proteins. For instance, using poly(ethylene glycol) (PEG) hydrogels with tethered small chemical functional groups, human MSCs were induced to differentiate towards osteogenic and adipogenic lineages.7 Interestingly, charged phosphate functional groups, prominent in mineralized bone, were responsible for osteogenic induction, whereas hydrophobic groups, reflecting the lipid environment of adipose tissue, resulted in adipogenic differentiation.
Synthetic biomaterials and microfabrication technologies have been combined to generate homogeneous populations of stem cells.59, 62, 92, 126 Cross-linkable polymers and photolithography can be used to construct PEG hydrogel microwells to generate a uniform array of controlled-size embryoid bodies (EBs).62, 92, 126 EBs are multicellular aggregates of ESCs that recapitulate the early steps in development. Hwang et al.59 used such a platform to control EB size based on the microwell dimension, and study its effects on ESC differentiation. Smaller EBs favored vascular differentiation mediated via expression of Wnt5a, whereas larger EBs promoted cardiomyogenesis through expression of Wnt11. Photolithography can also be used to create asymmetrical microenvironments to recapitulate in vivo developmental processes. Qi et al.108 encapsulated one-half of an EB in PEG hydrogel and the other half in gelatin methacrylate hydrogel. This resulted in polarization of EBs and vasculogenic differentiation in a spatially dependent manner.
In addition to polymers, inorganic materials, such as ceramics142 and metals78 have also been under investigation, primarily as osteogenic materials in MSC differentiation.
Creating Biomaterial Libraries
Given the vast number of ECM and synthetic molecules used as scaffolds and the difficulty in predicting the reaction of stem cells to such molecules, high-throughput screening is needed for an efficient and cost-effective method of creating material libraries.37, 69 Such screening can be done with the aid of robotically-assisted, nanoliter-scale liquid dispensing, and printing methods. Inone example, Flaim et al.38 created a 32 ECM combinatorial platform made from five ECM molecules (collagen I, III, IV, laminin, and fibronectin) and demonstrated that hepatocyte differentiation from mouse ESCs was enhanced ~140-fold between the least and the most effective combinations. In another work, arrays of self-assembled monolayers presenting varying peptide sequences and densities were used to screen for ESC growth and maintenance.26 An appropriate peptide sequence was chosen based on the screening results, and used to create a 3D hydrogel scaffold that, as expected, promoted ESC self-renewal. High-throughput platforms have also been applied to analyze the effects of combinatorial libraries of synthetic polymers on stem cells. For instance, Anderson et al.5 tested over 1,700 human ESC-acrylate-based polymer interactions using nanoliter quantities of materials to measure cellular attachment, proliferation, and differentiation (Fig. 2d). Using their microarray, materials that enhanced cell attachment, growth, and differentiation into cytokeratin-positive cells were identified.
ECM interaction with cells, mediated through cell membrane receptors, is both biochemical and biophysical in nature. The biophysical features experienced by stem cells include topography, matrix stiffness, and dynamic forces. These physical cues exerted on the cell by the microenvironment are transduced to biochemical intracellular signals and cytoskeletal tension through actomyosin contractility that result in cellular proliferation, apoptosis, adhesion, migration, or differentiation.40, 138 These interactions serve as a salient regulator of stem cell fate.30, 52
Topography
ECM is known to exhibit topographical cues at the nano- and micrometer scales that interact with cells. For instance, collagen and elastin have a fibrillar structure with nanometer dimensions. This interaction with cells is through a phenomenon known as contact guidance. Contact guidance is the process by which ECM provides directional cues that will determine cell morphology and migration.111 Nanoscale technologies have been used to create surface geometrical arrays of nanoposts, nanogrooves, and nanopits.8, 33, 71, 124, 136 Methods used to fabricate these topographical cues include traditional nanomolding, photolithography, electron-beam,131 and dip pen75 lithography.97 These arrays have been shown to induce cytoskeletal changes and commitment in stem cells. For instance, fibronectin-coated poly(dimethylsiloxane) (PDMS) nanometer line gratings resulted in alignment and elongation of human ESCs through the organization of actin, vimentin, and α-tubulin.46 Similarly, in work by Yim et al.141, human MSCs that were cultured on collagen-coated PDMS nanogratings showed increased cytoskeletal and nuclei alignment, as well as upregulation of microtubule-associated protein 2, a neuronal marker, compared to unpatterned and micropatterned controls (Fig. 3a). In work by Oh et al.98, human MSC differentiation was controlled by altering the size of the titanium oxide nanotubes on which cells were cultured. Notably, small diameter (~30-nm) nanotubes maintained multipotency while larger (~70- to 100-nm) nanotubes promoted differentiation to osteoblast-like cells in the absence of osteogenic inducing factors. In another work, Dalby et al.21 showed that human MSCs grown on slightly disordered polymethylmethacrylate nanopits resulted in the increased production of bone-specific extracellular matrices, osteopontin and osteocalcin, in the absence of osteogenic inducing media. There has not been a clear consensus on the mechanism explaining the observed cellular phenotypic changes due to topography. One theory states that such changes originate from the generation of anisotropic stresses that may be due to clustering of focal adhesions or directional actin polymerization.8 Although the exact mechanism is yet to be determined, topographical features add another degree of control over scaffold design for stem cell engineering.
Figure 3
Figure 3
Role of physical cues on stem cells. (a) Scanning electron micrographs of PDMS nano-patterns (top) and human MSCs on a nano-patterned substrate (middle) and F-actin immunostaining (bottom).141 Adapted with permission from Elsevier. (b) Cell shape directing (more ...)
Microarchitecture and Cell Shape
During embryonic development, cells change their shape as they undergo differentiation. Indeed, cell shape is known to affect proliferation,41 apoptosis,15 nuclear organization,73 and differentiation.44, 66, 88 There is an intricate link between cell shape and cell-ECM interaction, which is mediated through cytoskeletal changes.35 The local microarchitecture and geometry of the niche, imparted by ECM and neighboring cells, is one of the physical cues experienced by stem cells. Microfabrication techniques have allowed for the manipulation of cell shape, through creating cell-adhesive geometric patterns that in turn lead to changes in cell function.65 For example, McBeath et al.88 demonstrated that lineage commitment of stem cells can be controlled through cell morphology. In their study, human MSCs were grown on adhesive substrates that either allowed flat or round morphologies. Osteogenesis was favored in cells that adhered and spread, whereas adipogenesis was enhanced in cells that did not spread. Furthermore, these commitments were dependent on RhoA activity, known to be involved in cytoskeletal remodeling, and actomyosin tension. Using the same patterned substrates, human MSCs were selectively differentiated to myogenic (flattened morphology) or chondrogenic (round shape) fates in the presence of transforming growth factor β.44
To further elucidate the interwoven roles of cytoskeletal tension and cell shape on stem cell fate, in another study, geometrical patterns of varying aspect ratios and subcellular curvatures were tested on human MSCs (Fig. 3b).66 Shapes that had higher aspect ratios and greater curvatures promoted increased contractility and preferential osteogenesis, whereas shapes resulting in low contractility favored adipogenesis. The effect of cytoskeletal contractility on stem cell fate, mediated through cell shape, was confirmed by the use of cytoskeletal-disrupting agents.
The previous examples demonstrate that human MSCs can be, depending on the growth factors present, directed to osteogenic66, 88 and myogenic44 fates when cell spreading and increased contractility are favored, whereas adipogenic66, 88 and chondrogenic44 differentiation are enhanced when cell size and contractility are reduced. Further, these studies demonstrate the delicate interplay between cytoskeletal rearrangement, cell shape, and differentiation. Such an interplay may be explained by (1) changing cytoskeletal arrangement leading to a distorted nucleus;119 and (2) focal adhesion assembly induction.14
Substrate Stiffness and Dynamic Forces
Not only can the ECM influence the behavior of stem cells through its topographical/geometric features, its inherent mechanical properties add another dimension to the complexity of the stem cell niche and control over stem cell regulation. Materials science and polymer chemistry have been used to fabricate materials with tunable stiffness. One common method is by altering the degree of cross-linking of hydrogels.122 In a seminal work, Engler et al.34 used collagen I-coated polyacrylamide gels with elastic moduli resembling brain, muscle and bone tissues to induce the commitment of human MSCs into neurogenic, myogenic and osteogenic lineages, respectively. In the previous example, cells were grown in a 2D environment. To study the effects of matrix stiffness on stem cell fate in a more physiologically relevant environment, MSCs were encapsulated in 3D RGD-modified alginate hydrogels of varying elastic moduli.57 Osteogenesis was predominant in hydrogels with elastic moduli of 11–30 kPa, whereas adipogenesis occurred more in softer gels (2.5–5 kPa). While the aforementioned studies control adhesive ligand densities in their materials, other material properties, such as surface chemistry and porosity, cannot be effectively controlled in the fabrication process of materials with varying stiffness. To isolate the effect of substrate rigidity, PDMS microposts of different rigidities were fabricated by varying the post’s height (Figure 3c).42 Human MSCs grown on rigid (short) microposts favored osteogenesis whereas adipogenesis occurred on softer (tall) microposts. Substrate elasticity can also be used to direct the fate of other stem cells, such as hematopoietic,56 neural,114 and muscle.48
Whereas stem cell fate can be dictated by static forces through matrix stiffness, during organogenesis as well as adult life, stem cells are exposed to dynamic forces that also affect their function.1, 16 For instance, local cyclic stress applied through magnetic twisting cytometry to single ESCs resulted in the downregulation of pluripotency genes, Oct3/4, as compared to unexposed cells in the same culture dish.16 Moreover, shear forces from flowing fluids have been found to induce endothelial139 and hematopoietic lineage differentiation. For example, Adamo et al.1 described the preferential differentiation of mouse ESCs into hematopoietic progenitor cells when exposed to shear stress as opposed to static culture. Briefly, mouse ESCs, cultured on 1% gelatin-coated culture plates, were exposed to a step-wise increase in shear stress from 0–5 dyne per cm2, followed by a constant 5 dyne per cm2. Compared to static culture, the cells in dynamic culture had a higher expression of the endothelial and hematopoietic marker, CD31 (PECAM1).
In addition to the cellular scaffold and physical facets, biochemical factors are a vital component of the stem cell niche. These biochemical factors present themselves in a dynamic, spatiotemporal manner that is difficult to mimic in standard in vitro culture systems. Identifying relevant biomolecules involved in stem cell modulation is critical to understanding mechanisms of self-renewal and differentiation, as well as directing such processes.
Static Biochemical Presentation
In the stem cell niche, cytokines and other biochemical signals are released by stem cells (autocrine), support (paracrine), or distant cells (endocrine) that act on stem cells to induce functional changes. Cytokines can be incorporated into biomaterial scaffolds in soluble (interspersed), immobilized, or encapsulated forms. One of the most common methods of assimilating cytokines into scaffolds is by dispersing them in the interstices of the material. For instance, BMP-2 and human MSCs were added prior to gelation, and encapsulated in HA hydrogels to induce osteogenesis in rat calvarial defects.67 Hydrogels with human MSCs and BMP-2 had the most enhanced mature bone formation with vascular markers being present, compared with hydrogel alone, hydrogel and MSCs, or hydrogel and BMP-2.
While cytokines are part of the culture media in conventional culture systems, in vivo, they are often tethered to the ECM. To replicate this mode of presentation, cytokines or specific biologically-active sequences of cytokines (peptides) have been immobilized onto biomaterial scaffolds. For example, Alberti et al.3 immobilized leukemia inhibitory factor (LIF), conventionally used in ESC culture to inhibit differentiation, to maleic anhydride copolymer thin-film coatings, and showed that stem cell pluripotency could be maintained for at least two weeks in the absence of soluble LIF. Covalent bonding of proteins to scaffolds may undoubtedly raise concern over the structure and bio-functionality of the tethered protein. To overcome this concern, other biomimetic methods have been developed. Glycosaminoglycans (GAGs), as part of proteoglycans (a component of ECM), are known to bind growth factors via electrostatic interactions, leading to their sequestration and reduced degradation.107 Heparin, a well-known GAG, has been incorporated in scaffolds to immobilize growth factors and influence stem cells.103 Another method of incorporating cytokines is by encapsulating them into polymer-based nano- or microspheres embedded within scaffolds.54, 110 For instance, biodegradable poly(lactic-co-glycolic acid) microspheres loaded with retinoic acid (RA) were fabricated by water-in-oil single emulsion and incorporated in EBs.13 In EBs containing RA-loaded microspheres, ESC differentiation was more organized and better resembled early embryogenesis compared to EBs exposed to soluble RA in culture media. These delivery particles allowed for a controlled release and protected the biomolecule from inactivation.107
Miniaturized high-throughput platforms have also been extended to efficiently study stem cell-cytokine interactions. These assays have been employed to identify peptides26 and screen for small molecules,28 and combinations of ECM proteins and growth factors.39, 72, 123 For example, an array of combinations of ECM components and cytokines were printed onto a surface and seeded with human neural precursor cells. Self-renewal and proliferation were enhanced by the co-stimulation of Wnt and Notch, whereas BMP-4 induced differentiation into cells expressing neural and glial markers. These microarrays can be used to discover new interactions between stem cells and their soluble environment for the development of new pharmaceutical therapeutics. In addition, the large quantity of data obtained via these methods poses a challenge and necessitates a need for an accurate, efficient and automated computer-aided analysis.
Dynamic Biochemical Presentation
The stem cell niche in vivo, unlike conventional culture systems, is a dynamic milieu where biochemical cues are presented in a spatially and temporally distinct manner. The use of microfluidics has added valuable insight and allowed for generation of a more dynamic microenvironment for stem cell manipulation. Microfluidics is the science of manipulating fluids using channels with dimensions in the micrometer range.137 Currently, the most popular material used to fabricate microfluidic devices is PDMS, which is well suited for biological applications given its low toxicity, high transparency, and permeability to oxygen and carbon dioxide.130 Given that flow is laminar in such devices, mixing occurs via diffusion; hence, there is great control over the spatial and temporal concentrations of molecules (Fig. 4a). There are numerous methods of generating chemical concentration gradients that have been reviewed in the literature.63, 116
Figure 4
Figure 4
Microfluidics used for dynamic biochemical presentation. (a) A ‘Christmas-tree’ microfluidic device used to produce soluble concentration gradients.10 (Adapted with permission from American Chemical Society) (b) Microfluidic platform with (more ...)
The presentation of numerous soluble factors (e.g. cytokines and oxygen) and physical parameters (e.g. shear stress and temperature) can be controlled with microfluidics. By exposing a Drosophila melanogaster embryo to a temperature gradient along its anterior-posterior axis, Lucchetta et al.79 were able to generate a differential cellular proliferation within the embryo along the temperature gradient. The generation of chemical gradients via diffusive mixing, devised by Dertinger, Jeon, and co-workers, has been used extensively to study chemotaxis and other biological phenomena involving gradients.27, 60 Using a tree-like gradient-generating microfluidic system, Chung et al.17 studied the effects of a concentration gradient of a mixture of epidermal growth factor, FGF-2, and platelet-derived growth factor on the differentiation of human NSCs into astrocytes. One of the limitations of microfluidics has been the adverse effect of the shear stress on cells. Other methods of generating gradients, such as using an osmotic pump, have been introduced to reduce the magnitude of the shear stress produced. For instance, Park et al.102 used an osmotic pump to generate continuous gradients of cytokines (sonic hedgehog, FGF-8, and BMP-4) to direct the differentiation of ESC-derived neural progenitors into neurons.
Analysis of in vitro cultures of mixed cell colonies results in the average response of the entire population, masking the responses of individual or rare cells. Hence, there is a need for single cell culture and analysis platforms. Microfluidics have generated approaches to address this issue. Lecault et al.74 fabricated a multi-layered microfluidic device with nanoliter-size chambers and automated medium exchange to study HSC proliferation in response to Steel factor (Fig. 4b). Such a device allows for single-cell level analysis, continuous medium exchange, parallel studies of temporally-varied exposure of cells to cytokines (in this case, Steel factor), and time-lapse imaging.
Thus far, we have broken down the stem cell niche into its individual elements to better comprehend the role of each component in stem cell function. However, in order to materialize the potential benefits of stem cell engineering-driven therapeutics, construction of complex, hierarchical structures and tissues is necessary. Tissue printing is a promising technology for such 3D tissue fabrication.90, 91 Using a ‘bottom-up’ approach, tissue printing uses robotically-driven, layer-by-layer deposition of cells and matrices without the use of a scaffold (Fig. 5a).12 Although this technology has not been utilized widely in stem cell biology, it has the potential to make an impact. For instance, bioprinting has been used to create distinct spatial patterns of immobilized BMP-2 and FGF-2 that resulted in the commitment of primary muscle-derived stem cells to osteoblast and tenocyte lineages, respectively, when on-pattern and myogenic lineage when off-pattern.64
Figure 5
Figure 5
Engineering methods used to construct tissue-like structures. (a) 3D bioprinting schematic: layer-by-layer deposition of cell aggregates.127 (Adapted with permission from John Wiley and Sons) (b) Cell patterning and multicellular assembly by dielectrophoresis (more ...)
Another technology of interest is electropatterning for the construction of 3D microenvironments. Albrecht et al.4 used dielectrophoretic forces to form multicellular structures ranging from a few to >20,000 cell clusters in photopolymerizable hydrogels and demonstrated that chondrocyte synthesis of sulfated GAG was dependent upon cluster size (Fig. 5b). Another ‘bottom-up’ approach is cell-laden hydrogel modular assembly.61 In this method individual building blocks or microstructures of a tissue are built and assembled into a cohesive tissue-like structure (Fig. 5c). Each unit may contain a particular cell type and embody a scaffold with unique mechanical and biochemical properties. In this way, not only is the architecture of the desired tissue defined, but also the cellular arrangements can be meticulously delineated. In one example, Du et al.31 assembled tubular structures of concentric hydrogel constructs with an inner layer of encapsulated endothelial cells and outer layer of smooth muscle cells, emulating native arterial cellular organization.
Up until now, we have discussed the application of biomaterials for the in vitro study of stem cell biology. Biomaterials can also be used as delivery vehicles for stem cells in vivo for the repair of damaged or degenerated tissue.30, 81, 82, 93 Currently, most cell-based therapies involve injecting cells in a liquid medium into the site of interest. However, in this method of delivery, cells have low survivability due to anoikis, poor engraftment, and in the case of stem cells, control over differentiation is lacking.93 Advanced biomaterials have great potential in overcoming these shortcomings. These materials must be designed to create an environment for cell survival, provide support cells that function to enhance the endogenous stem cell population, and/or present regulatory cues that will modulate delivered or endogenous stem cells. Using this strategy, Hill et al.55 transplanted alginate scaffolds containing immobilized RGD, soluble hepatocyte growth factor and FGF-2, and satellite cells to damaged mouse muscle tissue. This resulted in enhanced activation and migration of transplanted cells and repopulation of damaged muscle tissue as compared with simply injecting satellite cells. In another study, Silva et al.120 showed that alginate scaffolds containing RGD and VEGF along with a combination of outgrowth endothelial cells and endothelial progenitor cells can salvage ischemic murine limbs and restore limb perfusion as compared to dual cell injection (Fig. 6).
Figure 6
Figure 6
Biomaterials as delivery vehicles for cells. Alginate scaffolds with RGD, VEGF, endothelial progenitor cells (EPC) and outgrowth endothelial cells (OEC) salvaged ischemic murine hind limbs as shown in gross and perfusion images.120 Copyright (2008) National (more ...)
While stem cell transplantation remains a viable option in tissue repair, therapeutic targeting of endogenous stem cell niches has been achieved via soluble cues and support cells.2, 19, 113 Smart biomaterials can be applied to present localized, bioactive molecular cues or support cells targeting specific stem cell niches in vivo.51, 112, 144 These molecular cues may include growth factors and morphogens that can be released in a controlled fashion and target desired niches to affect the proliferation and differentiation of cells. For instance, Gomi et al.49 were able to drive heterotopic hematopoiesis of host origin and osteogenesis in a subcutaneously implanted polyester scaffold seeded with osteogenic cells. Furthermore, by injecting self-assembling peptide nanofibers into the myocardium, Davis et al.23 showed vascular progenitor cell recruitment.
Undoubtedly, stem cell-based therapeutics have become a tantalizing approach to the problems facing regenerative medicine. Aging, disease, and trauma can often lead to loss of tissues and with it, its function necessary for a patient’s quality of living and survival. Given their differentiation capacity, stem cells are an attractive choice to replace lost tissues. Since isolation of human ESCs and genetic engineering of iPSCs have occurred only in recent history, the merger of stem cell biology and engineering approaches are still nascent. Current conventional approaches to stem cell biology employ materials and systems that do not appropriately capture the spatial and temporal biochemical and biophysical cues present in vivo. This has resulted in frustration over controlling stem cell fate and translating in vitro work to animal models and human clinical trials. Microengineering and materials science disciplines have come to illuminate the complexity of the stem cell niche through novel, physiologically-relevant culture platforms. Such mimicry through multiplexed systems is necessary to study stem cell biology and pave the way towards clinical relevance. While nano- and microtechnologies have enabled precise control over studying the effects of individual components of the stem cell niche, the integration of these different elements in tissue engineered constructs will be the focus of future research.
ACKNOWLEDGMENTS
The authors acknowledge funding from the National Science Foundation CAREER Award (DMR 0847287), the office of Naval Research Young National Investigator Award and the National Institutes of Health (HL092836, DE019024, EB008392, DE021468, AR05837, EB012597, HL099073).
Footnotes
CONFLICTS OF INTEREST
The authors declare no competing interests.
REFERENCES
1. Adamo L, Naveiras O, Wenzel PL, McKinney-Freeman S, Mack PJ, Gracia-Sancho J, Suchy-Dicey A, Yoshimoto M, Lensch MW, Yoder MC. Biomechanical forces promote embryonic haematopoiesis. Nature. 2009;459:1131–1135. [PMC free article] [PubMed]
2. Adams GB, Martin RP, Alley IR, Chabner KT, Cohen KS, Calvi LM, Kronenberg HM, Scadden DT. Therapeutic targeting of a stem cell niche. Nat. Biotechnol. 2007;25:238–243. [PubMed]
3. Alberti K, Davey RE, Onishi K, George S, Salchert K, Seib FP, Bornhäuser M, Pompe T, Nagy A, Werner C, Zandstra PW. Functional immobilization of signaling proteins enables control of stem cell fate. Nat. Methods. 2008;5:645–650. [PubMed]
4. Albrecht DR, Underhill GH, Wassermann TB, Sah RL, Bhatia SN. Probing the role of multicellular organization in three-dimensional microenvironments. Nat. Methods. 2006;3:369–375. [PubMed]
5. Anderson DG, Levenberg S, Langer R. Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells. Nat. Biotechnol. 2004;22:863–866. [PubMed]
6. Baker BM, Gee AO, Metter RB, Nathan AS, Marklein RA, Burdick JA, Mauck RL. The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers. Biomaterials. 2008;29:2348–2358. [PMC free article] [PubMed]
7. Benoit DS, Schwartz MP, Durney AR, Anseth KS. Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. Nat. Mater. 2008;7:816–823. [PMC free article] [PubMed]
8. Bettinger CJ, Langer R, Borenstein JT. Engineering substrate topography at the micro-and nanoscale to control cell function. Angew. Chem. Int. Edit. 2009;48:5406–5415. [PMC free article] [PubMed]
9. Bhardwaj G, Murdoch B, Wu D, Baker DP, Williams KP, Chadwick K, Ling LE, Karanu FN, Bhatia M. Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. Nat. Immunol. 2001;2:172–180. [PubMed]
10. Burdick JA, Khademhosseini A, Langer R. Fabrication of gradient hydrogels using a microfluidics/photopolymerization process. Langmuir. 2004;20:5153–5156. [PubMed]
11. Burdick JA, Vunjak-Novakovic G. Engineered microenvironments for controlled stem cell differentiation. Tissue Eng Part A. 2009;15:205–219. [PMC free article] [PubMed]
12. Campbell PG, Weiss LE. Tissue engineering with the aid of inkjet printers. Expert Opin. Biol. Th. 2007;7:1123–1127. [PubMed]
13. Carpenedo RL, Bratt-Leal AM, Marklein RA, Seaman SA, Bowen NJ, McDonald JF, McDevitt TC. Homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules. Biomaterials. 2009;30:2507–2515. [PMC free article] [PubMed]
14. Chen CS, Alonso JL, Ostuni E, Whitesides GM, Ingber DE. Cell shape provides global control of focal adhesion assembly. Biochem. Biophys. Res. Commun. 2003;307:355–361. [PubMed]
15. Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science. 1997;276:1425–1428. [PubMed]
16. Chowdhury F, Na S, Li D, Poh YC, Tanaka TS, Wang F, Wang N. Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cells. Nat. Mater. 2010;9:82–88. [PMC free article] [PubMed]
17. Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL. Human neural stem cell growth and differentiation in a gradient-generating microfluidic device. Lab Chip. 2005;5:401–406. [PubMed]
18. Clevers H. The cancer stem cell: premises, promises and challenges. Nat. Med. 2011;17:313–319. [PubMed]
19. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature. 2005;433:760–764. [PubMed]
20. Connelly JT, Gautrot JE, Trappmann B, Tan DWM, Donati G, Huck WTS, Watt FM. Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions. Nat. Cell Biol. 2010;12:711–718. [PubMed]
21. Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO, Herzyk P, Wilkinson CDW, Oreffo ROC. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat. Mater. 2007;6:997–1003. [PubMed]
22. Daley GQ, Scadden DT. Prospects for stem cell-based therapy. Cell. 2008;132:544–548. [PubMed]
23. Davis ME, Motion JPM, Narmoneva DA, Takahashi T, Hakuno D, Kamm RD, Zhang S, Lee RT. Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation. 2005;111:442–450. [PMC free article] [PubMed]
24. Dawson E, Mapili G, Erickson K, Taqvi S, Roy K. Biomaterials for stem cell differentiation. Adv. Drug Delivery Rev. 2008;60:215–228. [PubMed]
25. de Haan G, Weersing E, Dontje B, van Os R, Bystrykh LV, Vellenga E, Miller G. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Dev. Cell. 2003;4:241–251. [PubMed]
26. Derda R, Li L, Orner BP, Lewis RL, Thomson JA, Kiessling LL. Defined substrates for human embryonic stem cell growth identified from surface arrays. ACS Chem. Biol. 2007;2:347–355. [PubMed]
27. Dertinger SKW, Chiu DT, Jeon NL, Whitesides GM. Generation of gradients having complex shapes using microfluidic networks. Anal. Chem. 2001;73:1240–1246.
28. Desbordes SC, Placantonakis DG, Ciro A, Socci ND, Lee G, Djaballah H, Studer L. High-throughput screening assay for the identification of compounds regulating self-renewal and differentiation in human embryonic stem cells. Cell Stem Cell. 2008;2:602–612. [PMC free article] [PubMed]
29. Dickinson LE, Kusuma S, Gerecht S. Reconstructing the differentiation niche of embryonic stem cells using biomaterials. Macromol. Biosci. 2011;11:36–49. [PubMed]
30. Discher DE, Mooney DJ, Zandstra PW. Growth factors, matrices, and forces combine and control stem cells. Science. 2009;324:1673–1677. [PMC free article] [PubMed]
31. Du Y, Ghodousi M, Qi H, Haas N, Xiao W, Khademhosseini A. Sequential assembly of cell-laden hydrogel constructs to engineer vascular-like microchannels. Biotechnol. Bioeng. 2011;108:1693–1703. [PMC free article] [PubMed]
32. Du Y, Lo E, Ali S, Khademhosseini A. Directed assembly of cell-laden microgels for fabrication of 3D tissue constructs. Proc. Natl. Acad. Sci. USA. 2008;105:9522–9527. [PubMed]
33. Dvir T, Timko BP, Kohane DS, Langer R. Nanotechnological strategies for engineering complex tissues. Nat. Nanotechnol. 2010;6:13–22. [PubMed]
34. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126:677–689. [PubMed]
35. Eyckmans J, Boudou T, Yu X, Chen CS. A hitchhiker's guide to mechanobiology. Dev. Cell. 2011;21:35–47. [PMC free article] [PubMed]
36. Fan VH, Au A, Tamama K, Littrell R, Richardson LB, Wright JW, Wells A, Griffith LG. Tethered epidermal growth factor provides a survival advantage to mesenchymal stem cells. Stem Cells. 2007;25:1241–1251. [PubMed]
37. Fernandes TG, Diogo MM, Clark DS, Dordick JS, Cabral J. High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research. Trends Biotechnol. 2009;27:342–349. [PubMed]
38. Flaim CJ, Chien S, Bhatia SN. An extracellular matrix microarray for probing cellular differentiation. Nat. Methods. 2005;2:119–125. [PubMed]
39. Flaim CJ, Teng D, Chien S, Bhatia SN. Combinatorial signaling microenvironments for studying stem cell fate. Stem Cells Dev. 2008;17:29–40. [PubMed]
40. Fletcher DA, Mullins RD. Cell mechanics and the cytoskeleton. Nature. 2010;463:485–492. [PMC free article] [PubMed]
41. Folkman J, Moscona A. Role of cell shape in growth control. Nature. 1978;273:345–349. [PubMed]
42. Fu J, Wang YK, Yang MT, Desai RA, Yu X, Liu Z, Chen CS. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods. 2010;7:733–736. [PMC free article] [PubMed]
43. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their niche. Cell. 2004;116:769–778. [PubMed]
44. Gao L, McBeath R, Chen CS. Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-Cadherin. Stem Cells. 2010;28:564–572. [PMC free article] [PubMed]
45. Geckil H, Xu F, Zhang X, Moon SJ, Demirci U. Engineering hydrogels as extracellular matrix mimics. Nanomedicine. 2010;5:469–484. [PMC free article] [PubMed]
46. Gerecht S, Bettinger CJ, Zhang Z, Borenstein JT, Vunjak-Novakovic G, Langer R. The effect of actin disrupting agents on contact guidance of human embryonic stem cells. Biomaterials. 2007;28:4068–4077. [PMC free article] [PubMed]
47. Gerecht S, Burdick JA, Ferreira LS, Townsend SA, Langer R, Vunjak-Novakovic G. Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells. Proc. Natl. Acad. Sci. USA. 2007;104:11298–11303. [PubMed]
48. Gilbert PM, Havenstrite KL, Magnusson KEG, Sacco A, Leonardi NA, Kraft P, Nguyen NK, Thrun S, Lutolf MP, Blau HM. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science. 2010;329:1078–1081. [PMC free article] [PubMed]
49. Gomi K, Kanazashi M, Lickorish D, Arai T, Davies JE. Bone marrow genesis after subcutaneous delivery of rat osteogenic cell-seeded biodegradable scaffolds into nude mice. J. Biomed. Mater. Res. A. 2004;71:602–607. [PubMed]
50. Grayson WL, Fröhlich M, Yeager K, Bhumiratana S, Chan M, Cannizzaro C, Wan LQ, Liu XS, Guo XE, Vunjak-Novakovic G. Engineering anatomically shaped human bone grafts. Proc. Natl. Acad. Sci. USA. 2010;107:3299–3304. [PubMed]
51. Gu F, Zhang L, Teply BA, Mann N, Wang A, Radovic-Moreno AF, Langer R, Farokhzad OC. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc. Natl. Acad. Sci. USA. 2008;105:2586–2591. [PubMed]
52. Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, Chen CS. Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell. 2009;5:17–26. [PMC free article] [PubMed]
53. Hahn MS, Miller JS, West JL. Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Adv. Mater. 2006;18:2679–2684.
54. Hanson JA, Chang CB, Graves SM, Li Z, Mason TG, Deming TJ. Nanoscale double emulsions stabilized by single-component block copolypeptides. Nature. 2008;455:85–88. [PubMed]
55. Hill E, Boontheekul T, Mooney DJ. Regulating activation of transplanted cells controls tissue regeneration. Proc. Natl. Acad. Sci. USA. 2006;103:2494–2499. [PubMed]
56. Holst J, Watson S, Lord MS, Eamegdool SS, Bax DV, Nivison-Smith LB, Kondyurin A, Ma L, Oberhauser AF, Weiss AS, Rasko JEJ. Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. Nat. Biotechnol. 2010;28:1123–1128. [PubMed]
57. Huebsch N, Arany PR, Mao AS, Shvartsman D, Ali OA, Bencherif SA, Rivera-Feliciano J, Mooney DJ. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 2010;9:518–526. [PMC free article] [PubMed]
58. Hwang NS, Varghese S, Zhang Z, Elisseeff J. Chondrogenic differentiation of human embryonic stem cell-derived cells in arginine-glycine-aspartate-modified hydrogels. Tissue Eng. 2006;12:2695–2706. [PubMed]
59. Hwang YS, Chung BG, Ortmann D, Hattori N, Moeller HC, Khademhosseini A. Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11. Proc. Natl. Acad. Sci. USA. 2009;106:16978–16983. [PubMed]
60. Jeon NL, Dertinger SKW, Chiu DT, Choi IS, Stroock AD, Whitesides GM. Generation of solution and surface gradients using microfluidic systems. Langmuir. 2000;16:8311–8316.
61. Kachouie NN, Du Y, Bae H, Khabiry M, Ahari AF, Zamanian B, Fukuda J, Khademhosseini A. Directed assembly of cell-laden hydrogels for engineering functional tissues. Organogenesis. 2010;6:234–244. [PMC free article] [PubMed]
62. Karp JM, Yeh J, Eng G, Fukuda J, Blumling J, Suh KY, Cheng J, Mahdavi A, Borenstein J, Langer R, Khademhosseini A. Controlling size, shape and homogeneity of embryoid bodies using poly (ethylene glycol) microwells. Lab Chip. 2007;7:786–794. [PubMed]
63. Keenan TM, Folch A. Biomolecular gradients in cell culture systems. Lab Chip. 2008;8:34–57. [PubMed]
64. Ker EDF, Chu B, Phillippi JA, Gharaibeh B, Huard J, Weiss LE, Campbell PG. Engineering spatial control of multiple differentiation fates within a stem cell population. Biomaterials. 2011;32:3413–3422. [PMC free article] [PubMed]
65. Khademhosseini A, Langer R, Borenstein J, Vacanti JP. Microscale technologies for tissue engineering and biology. Proc. Natl. Acad. Sci. USA. 2006;103:2480–2487. [PubMed]
66. Kilian KA, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc. Natl. Acad. Sci. USA. 2010;107:4872–4877. [PubMed]
67. Kim J, Kim IS, Cho TH, Lee KB, Hwang SJ, Tae G, Noh I, Lee SH, Park Y, Sun K. Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells. Biomaterials. 2007;28:1830–1837. [PubMed]
68. Kim K, Dean D, Lu A, Mikos AG, Fisher JP. Early osteogenic signal expression of rat bone marrow stromal cells is influenced by both hydroxyapatite nanoparticle content and initial cell seeding density in biodegradable nanocomposite scaffolds. Acta Biomater. 2011;7:1249–1264. [PMC free article] [PubMed]
69. Kobel S, Lutolf M. High-throughput methods to define complex stem cell niches. BioTechniques. 2010;48:ix–xxii. [PubMed]
70. Koh WG, Revzin A, Pishko MV. Poly (ethylene glycol) hydrogel microstructures encapsulating living cells. Langmuir. 2002;18:2459–2462. [PubMed]
71. Kulangara K, Leong KW. Substrate topography shapes cell function. Soft Matter. 2009;5:4072–4076.
72. LaBarge MA, Nelson CM, Villadsen R, Fridriksdottir A, Ruth JR, Stampfer MR, Petersen OW, Bissell MJ. Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments. Integr. Biol. 2009;1:70–79. [PMC free article] [PubMed]
73. Le Beyec J, Xu R, Lee SY, Nelson CM, Rizki A, Alcaraz J, Bissell MJ. Cell shape regulates global histone acetylation in human mammary epithelial cells. Exp. Cell Res. 2007;313:3066–3075. [PMC free article] [PubMed]
74. Lecault V, VanInsberghe M, Sekulovic S, Knapp DJHF, Wohrer S, Bowden W, Viel F, McLaughlin T, Jarandehei A, Miller M, Falconnet D, White AK, Kent DG, Copley MR, Taghipour F, Eaves CJ, Humphries RK, Piret JM, Hansen CL. High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays. Nat. Methods. 2011;8:581–586. [PubMed]
75. Lee KB, Park SJ, Mirkin CA, Smith JC, Mrksich M. Protein nanoarrays generated by dip-pen nanolithography. Science. 2002;295:1702–1705. [PubMed]
76. Levenberg S, Huang NF, Lavik E, Rogers AB, Itskovitz-Eldor J, Langer R. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc. Natl. Acad. Sci. USA. 2003;100:12741–12746. [PubMed]
77. Lin S, Sangaj N, Razafiarison T, Zhang C, Varghese S. Influence of Physical Properties of Biomaterials on Cellular Behavior. Pharm. Res. 2011;28:1422–1430. [PMC free article] [PubMed]
78. Lopez-Heredia MA, Sohier J, Gaillard C, Quillard S, Dorget M, Layrolle P. Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering. Biomaterials. 2008;29:2608–2615. [PubMed]
79. Lucchetta EM, Lee JH, Fu LA, Patel NH, Ismagilov RF. Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics. Nature. 2005;434:1134–1138. [PMC free article] [PubMed]
80. Lutolf MP. Integration column: artificial ECM: expanding the cell biology toolbox in 3D. Integr. Biol. 2009;1:235–241. [PubMed]
81. Lutolf MP, Blau HM. Artificial stem cell niches. Adv. Mater. 2009;21:3255–3268. [PMC free article] [PubMed]
82. Lutolf MP, Gilbert PM, Blau HM. Designing materials to direct stem-cell fate. Nature. 2009;462:433–441. [PMC free article] [PubMed]
83. Lutolf MP, Hubbell JA. Synthesis and physicochemical characterization of end-linked poly (ethylene glycol)-co-peptide hydrogels formed by Michael-type addition. Biomacromolecules. 2003;4:713–722. [PubMed]
84. Lutolf MP, Weber FE, Schmoekel HG, Schense JC, Kohler T, Muller R, Hubbell JA. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat. Biotechnol. 2003;21:513–518. [PubMed]
85. Lutz JF, Zarafshani Z. Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using azide-alkyne. Adv. Drug Delivery Rev. 2008;60:958–970. [PubMed]
86. Marklein RA, Burdick JA. Controlling stem cell fate with material design. Adv. Mater. 2010;22:175–189. [PubMed]
87. Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromolecules. 2002;3:232–238. [PubMed]
88. McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell. 2004;6:483–495. [PubMed]
89. Miller FD, Gauthier-Fisher A. Home at last: neural stem cell niches defined. Cell stem cell. 2009;4:507–510. [PubMed]
90. Mironov V, Boland T, Trusk T, Forgacs G, Markwald RR. Organ printing: computer-aided jet-based 3D tissue engineering. Trends Biotechnol. 2003;21:157–161. [PubMed]
91. Mironov V, Kasyanov V, Markwald RR. Organ printing: from bioprinter to organ biofabrication line. Curr. Opin. Biotech. 2011;22:1–7. [PubMed]
92. Moeller HC, Mian MK, Shrivastava S, Chung BG, Khademhosseini A. A microwell array system for stem cell culture. Biomaterials. 2008;29:752–763. [PMC free article] [PubMed]
93. Mooney DJ, Vandenburgh H. Cell delivery mechanisms for tissue repair. Cell Stem Cell. 2008;2:205–213. [PubMed]
94. Moore KA, Lemischka IR. Stem cells and their niches. Science. 2006;311:1880–1885. [PubMed]
95. Morrison SJ, Spradling AC. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell. 2008;132:598–611. [PubMed]
96. Ngai T, Behrens SH, Auweter H. Novel emulsions stabilized by pH and temperature sensitive microgels. Chem. Commun. 2005:331–333. [PubMed]
97. Norman JJ, Desai TA. Methods for fabrication of nanoscale topography for tissue engineering scaffolds. Ann. Biomed. Eng. 2006;34:89–101. [PubMed]
98. Oh S, Brammer KS, Li YSJ, Teng D, Engler AJ, Chien S, Jin S. Stem cell fate dictated solely by altered nanotube dimension. Proc. Natl. Acad. Sci. USA. 2009;106:2130–2135. [PubMed]
99. Orlando G, Wood KJ, Stratta RJ, Yoo JJ, Atala A, Soker S. Regenerative medicine and organ transplantation: past, present, and future. Transplantation. 2011;91:1310–1317. [PubMed]
100. Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, Taylor DA. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nat. Med. 2008;14:213–221. [PubMed]
101. Panda P, Ali S, Lo E, Chung BG, Hatton TA, Khademhosseini A, Doyle PS. Stop-flow lithography to generate cell-laden microgel particles. Lab Chip. 2008;8:1056–1061. [PMC free article] [PubMed]
102. Park JY, Kim SK, Woo DH, Lee EJ, Kim JH, Lee SH. Differentiation of neural progenitor cells in a microfluidic chip-generated cytokine gradient. Stem Cells. 2009;27:2646–2654. [PubMed]
103. Park K, Cho KJ, Kim JJ, Kim IH, Han DK. Functional PLGA scaffolds for chondrogenesis of bone-marrow-derived mesenchymal stem cells. Macromolecular bioscience. 2009;9:221–229. [PubMed]
104. Patterson J, Hubbell JA. Enhanced proteolytic degradation of molecularly engineered PEG hydrogels in response to MMP-1 and MMP-2. Biomaterials. 2010;31:7836–7845. [PubMed]
105. Peerani R, Rao BM, Bauwens C, Yin T, Wood GA, Nagy A, Kumacheva E, Zandstra PW. Niche-mediated control of human embryonic stem cell self-renewal and differentiation. EMBO J. 2007;26:4744–4755. [PubMed]
106. Petersen TH, Calle EA, Zhao L, Lee EJ, Gui L, Raredon MSB, Gavrilov K, Yi T, Zhuang ZW, Breuer C, Herzog E, Niklason LE. Tissue-engineered lungs for in vivo implantation. Science. 2010;329:538–541. [PubMed]
107. Place ES, Evans ND, Stevens MM. Complexity in biomaterials for tissue engineering. Nat. Mater. 2009;8:457–470. [PubMed]
108. Qi H, Du Y, Wang L, Kaji H, Bae H, Khademhosseini A. Patterned differentiation of individual embryoid bodies in spatially organized 3D hybrid microgels. Adv. Mater. 2010;22:5276–5281. [PMC free article] [PubMed]
109. Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K, Hintz L, Nusse R, Weissman IL. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature. 2003;423:409–414. [PubMed]
110. Richardson TP, Peters MC, Ennett AB, Mooney DJ. Polymeric system for dual growth factor delivery. Nat. Biotechnol. 2001;19:1029–1034. [PubMed]
111. Rorth P. Whence directionality: guidance mechanisms in solitary and collective cell migration. Dev. Cell. 2011;20:9–18. [PubMed]
112. Rothenfluh DA, Bermudez H, O’Neil CP, Hubbell JA. Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage. Nat. Mater. 2008;7:248–254. [PubMed]
113. Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, Tagliafico E, Ferrari S, Robey PG, Riminucci M, Bianco P. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell. 2007;131:324–336. [PubMed]
114. Saha K, Keung AJ, Irwin EF, Li Y, Little L, Schaffer DV, Healy KE. Substrate modulus directs neural stem cell behavior. Biophys. J. 2008;95:4426–4438. [PubMed]
115. Saha K, Pollock JF, Schaffer DV, Healy KE. Designing synthetic materials to control stem cell phenotype. Curr. Opin. Chem. Biol. 2007;11:381–387. [PMC free article] [PubMed]
116. Sant S, Hancock MJ, Donnelly JP, Iyer D, Khademhosseini A. Biomimetic Gradient Hydrogels for Tissue Engineering. Can. J. Chem. Eng. 2010;88:899–911. [PMC free article] [PubMed]
117. Scadden DT. The stem-cell niche as an entity of action. Nature. 2006;441:1075–1079. [PubMed]
118. Shastri VP, Martin I, Langer R. Macroporous polymer foams by hydrocarbon templating. Proc. Natl. Acad. Sci. USA. 2000;97:1970–1975. [PubMed]
119. Shivashankar GV. Mechanosignaling to the cell nucleus and gene regulation. Ann. Rev. Biophys. 2011;40:361–378. [PubMed]
120. Silva EA, Kim ES, Kong HJ, Mooney DJ. Material-based deployment enhances efficacy of endothelial progenitor cells. Proc. Natl. Acad. Sci. USA. 2008;105:14347–14352. [PubMed]
121. Skelley AM, Kirak O, Suh H, Jaenisch R, Voldman J. Microfluidic control of cell pairing and fusion. Nat. Methods. 2009;6:147–152. [PMC free article] [PubMed]
122. Slaughter BV, Khurshid SS, Fisher OZ, Khademhosseini A, Peppas NA. Hydrogels in regenerative medicine. Adv. Mater. 2009;21:3307–3329. [PubMed]
123. Soen Y, Mori A, Palmer TD, Brown PO. Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments. Mol. Syst. Biol. 2006;2:1–14. [PMC free article] [PubMed]
124. Stevens MM, George JH. Exploring and engineering the cell surface interface. Science. 2005;310:1135–1138. [PubMed]
125. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. [PubMed]
126. Tekin H, Anaya M, Brigham MD, Nauman C, Langer R, Khademhosseini A. Stimuli-responsive microwells for formation and retrieval of cell aggregates. Lab Chip. 2010;10:2411–2418. [PMC free article] [PubMed]
127. Thompson RP, Reckova M, de Almeida A, Bigelow MR, Stanley CP, Spruill JB, Trusk TT, Sedmera D. The oldest, toughest cells in the heart. In: Chadwick DJ, Goode J, editors. Development of the Cardiac Pacemaking and Conduction System. Chichester, UK: Wiley Online Library; 2003. pp. 157–176.
128. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. [PubMed]
129. Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, Shulman C, Milwid J, Kobayashi N, Tilles A, Berthiaume F, Hertl M, Nahmias Y, Yarmush ML, Uygun K. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat. Med. 2010;16:814–820. [PMC free article] [PubMed]
130. Van Noort D, Ong SM, Zhang C, Zhang S, Arooz T, Yu H. Stem cells in microfluidics. Biotechnol. Progr. 2009;25:52–60. [PubMed]
131. Vieu C, Carcenac F, Pepin A, Chen Y, Mejias M, Lebib A, Manin-Ferlazzo L, Couraud L, Launois H. Electron beam lithography: resolution limits and applications. Appl. Surf. Sci. 2000;164:111–117.
132. Voog J, Jones DL. Stem cells and the niche: a dynamic duo. Cell Stem Cell. 2010;6:103–115. [PMC free article] [PubMed]
133. Vunjak-Novakovic G, Scadden DT. Biomimetic platforms for human stem cell research. Cell Stem Cell. 2011;8:252–261. [PMC free article] [PubMed]
134. Ward JH, Peppas NA. Kinetic gelation modeling of controlled radical polymerizations. Macromolecules. 2000;33:5137–5142.
135. Wheeldon I, Ahari AF, Khademhosseini A. Microengineering Hydrogels for Stem Cell Bioengineering and Tissue Regeneration. J. Assoc. Lab. Autom. 2010;15:440–448. [PMC free article] [PubMed]
136. Wheeldon I, Farhadi A, Bick AG, Jabbari E, Khademhosseini A. Nanoscale tissue engineering: spatial control over cell-materials interactions. Nanotechnology. 2011;22 212001. [PMC free article] [PubMed]
137. Whitesides GM. The origins and the future of microfluidics. Nature. 2006;442:368–373. [PubMed]
138. Wozniak MA, Chen CS. Mechanotransduction in development: a growing role for contractility. Nat. Rev. Mol. Cell Biol. 2009;10:34–43. [PMC free article] [PubMed]
139. Yamamoto K, Sokabe T, Watabe T, Miyazono K, Yamashita JK, Obi S, Ohura N, Matsushita A, Kamiya A, Ando J. Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro. Am. J. Physiol. Heart Circ. Physiol. 2005;288:H1915–H1924. [PubMed]
140. Yeh J, Ling Y, Karp JM, Gantz J, Chandawarkar A, Eng G, Blumling J, III, Langer R, Khademhosseini A. Micromolding of shape-controlled, harvestable cell-laden hydrogels. Biomaterials. 2006;27:5391–5398. [PubMed]
141. Yim EKF, Pang SW, Leong KW. Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage. Exp. Cell Res. 2007;313:1820–1829. [PMC free article] [PubMed]
142. Yoshikawa H, Tamai N, Murase T, Myoui A. Interconnected porous hydroxyapatite ceramics for bone tissue engineering. J. R. Soc., Interface. 2009;6:S341–S348. [PMC free article] [PubMed]
143. Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, Ross J, Haug J, Johnson T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Linheng L. Identification of the haematopoietic stem cell niche and control of the niche size. Nature. 2003;425:836–841. [PubMed]
144. Zhao X, Kim J, Cezar CA, Huebsch N, Lee K, Bouhadir K, Mooney DJ. Active scaffolds for on-demand drug and cell delivery. Proc. Natl. Acad. Sci. USA. 2010;108:67–72. [PubMed]