Chapter 76

Designing Tunable Artificial Matrices forStem Cell Culture

Elizabeth F. Irwin,* Jacob F. Pollock,* David V. Schaffery,** and Kevin E. Healy*,z*Department of Bioengineering, yDepartment of Chemical Engineering, **The Helen Wills Neuroscience Institute, zDepartment of Materials Science and

Engineering, University of California at Berkeley, Berkeley, CA, USA

Ha

Co

Chapter Outline

Introduction 927

The Extracellular Matrix 928

Physical Properties of the Extracellular Matrix 928

Cell Adhesion and Mechanotransduction 928

Mechanics of the Natural ECM 929

Developing Artificial Matrices with Tunable Moduli for

Stem Cell Culture 929

Physical Structure of the Matrix 929

Choice of Material 929

ndbook of

pyright �

Natural Materials

929

Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00076-7

2013 Elsevier Inc. All rights reserved.

Synthetic Materials

930

Synthetic Materials with Bioactive Ligands

930

Creating Matrices with Tunable Moduli

931

Characterization of Matrix Mechanics 932

Atomic Force Microscopy 932

Rheology 932

Role of Matrix Mechanics in Stem Cell Behavior 933

Conclusions and Future Directions 934

References 934

INTRODUCTION

Embryonic stem (ES) cells, induced pluripotent stem (iPS)cells, and adult stem cells can generate a myriad of differentcells types in the body and thus have enormous potential foruse in regenerative medicine. In vivo, the fate of these stemcells, that is, to remain undifferentiated or to differentiateinto a particular cell type, is determined in large part bytheir local microenvironment, where the regulatorysignaling mechanisms include cellecell interactions;cellematrix interactions; growth factors; cytokines; and thephysiochemical nature of the environment, including theoxygen tension, osmolarity, and pH. The local microenvi-ronment, however, is highly dynamic, not only as organ-ismal development progresses, but also within thefluctuating nature of adult tissue.

Adult stem cells grow in niches and are often main-tained in a dormant, multipotent state where they retain theability to either self-renew or divide. These cells receivesignals through a diverse population of neighboringdifferentiated cell types, which secrete growth factors andcytokines and organize the extracellular matrix (Fuchset al., 2004). Niche cells thereby provide an environmentthat isolates stem cells from differentiation stimuli,

apoptotic stimuli, and excessive stem cell proliferation thatcould lead to cancer (Moore and Lemischka, 2006).However, with tissue injury and other processes associatedwith tissue turnover, the surrounding microenvironmentactively signals to these adult stem cells to begin toproliferate, self-renew, and/or differentiate to form newtissues.

The fate of the inner cell mass, the in vivo precursors ofES cells, is likewise determined by a complex sequence ofsignaling from the local environment, which in this caseprovides a more dynamic set of chemical and mechanicalsignals that orchestrate tissue formation and differentiation.During the earliest stages of this process, inner cell massconstituents interact with the matrix as it guides funda-mental processes of development including migration inthe early embryo, and the modulation of growth anddifferentiation programs of cells (Zagris, 2001). Subse-quently, as groups of cells form tissues, they experience notonly morphogen patterns but also tension, compression,and shear forces, and these mechanical forces can regulatethe expression of various genes (Brouzes and Farge, 2004).

In order to grow stem cells and tissues in vitro, it isnecessary to understand and attempt to reproduce the

927

928 VOLUME | 2 Adult and Fetal Stem Cells

complex microenvironment presented to these cells in vivo;however, current culture technologies are not sufficient tomimic this dynamic and intricate natural environment. Thischapter focuses on progress in the design and character-ization of artificial matrices that attempt to recapitulatemicroenvironmental cues for in vitro stem cell culture anddifferentiation.

THE EXTRACELLULAR MATRIX

Physical Properties of the ExtracellularMatrix

The natural extracellular matrix (ECM) provides a networkof chemically and physically associated proteins andpolysaccharides that allow cells to attach, migrate, andproliferate and also presents biochemical and physicalsignals affecting cell fate (Roskelley et al., 1995).A schematic of these interactions is shown in Figure 76.1.In addition, the ECM is not a static entity but insteadprovides a very dynamic environment whose componentsare locally secreted and restructured by cells. For example,as they move through the matrix, cells deposit new proteinsas well as locally cleave proteins by releasing metal-loproteinases (Streuli, 1999). In addition, the remodeling ofthe ECM is even more rapid in developing tissues, a processparticularly relevant to embryonic and fetal stem cells(Zagris, 2001).

Collagen scaffolds, which have been extensivelystudied, exemplify the basic architecture of the ECM. Intissues such as bone, cartilage, and tendons, collagen isarranged in fibrils to provide tensile strength. In contrast, inepithelial tissue, collagen forms a network of fibers asa basement membrane (Bosman and Stamenkovic, 2003),

FIGURE 76.1 Schematic of the mechanical interaction between a cell

and the surrounding ECM. Integrin receptors engage binding sites on

structural ECM proteins, bridging the cytoskeleton of the cell with the

surrounding matrix. Integrin binding at the surface can influence structural

rearrangements in both the cytoplasm and the nucleus.

an open structure that allows rapid diffusion of nutrients,metabolites, and hormones between the blood andconstituent cells. In addition to these two structures, there ishuge variability in collagen structure from tissue to tissue,as their complex architectures are composed of more than28 genetically distinct collagen molecules (Martin et al.,1985; Gordon and Hahn, 2010).

In addition to the collagen network in the ECM, thereexists long chain glycoaminoglycans (GAGs) and adhesiveproteins, including fibronectin, tenascin, and laminin.GAGs are highly hydrated and provide some compressivestrength to the network. Adhesive proteins present animmense number of physically immobilized and non-immobilized signals to the cells. Fibronectin, for example,is an important protein in guiding cell attachment andmigration during embryonic development, where itsabsence leads to defects in mesodermal, neural tube, andvascular development. Similarly, laminin has been shownto affect cell migration and differentiation in numeroussystems (Kubota et al., 1988).

Cell Adhesion and Mechanotransduction

Cell adhesion to theECM is crucial for both development andtissue maintenance (de Arcangelis and Georges-Labouesse,2000). Cell adhesion events mediate cell spreading, migra-tion, neurite extension, muscle-cell contraction, cell-cycleprogression, and differentiation (Giancotti and Ruoslahti,1999). Cells adhere to distinct adhesion domains on the ECM(Ruoslahti and Pierschbacher, 1987) through cell-surfacereceptors, primarily from the integrin family (Hynes, 2002).Upon engagement, these receptors provide chemical andmechanical signals to the cell that can lead to altered geneexpression and in some cases cell fate, including apoptosis,migration, differentiation, and proliferation.

Integrins are a family of approximately 25 membrane-spanning heterodimeric proteins containing ligand-bindingregions on the outer-membrane region and microfilaments-docking domains within the ectodomain. They arecomposed of a (~120 kD) and b subunits (~180 kD), andeach combination has a different binding affinity andsignaling properties. Most integrins are expressed on a widevariety of cell types, and most cells express several integrinreceptors.

Integrins signal both from the outside-in (binding of theintegrin with the ECM induces intracellular signalingevents) and the inside-out (the binding activity andexpression of integrins is regulated by the cell) (Giancottiand Ruoslahti, 1999). Following activation (via a bindingevent on the cytoplasmic domain), the focal adhesioncomplexes (FACs) bind actin-associated proteins such astalin, vinculin, zyxin, and paxillin and provide a directphysical link to the cytoskeleton, which also links to thenuclear scaffold. Integrin binding at the surface can

929Chapter | 76 Designing Tunable Artificial Matrices for Stem Cell Culture

therefore influence structural rearrangements in both thecytoplasm and the nucleus (Geiger et al., 2001). This isparticularly relevant since mechanical signals can poten-tially travel faster than signals that are mediated viadiffusion either across or through the cell.

Once bound to the ECM, integrins enable cells to sensethe physical and mechanical properties of the matrix. Asa result, changes in physical or mechanical properties of thematrix can activate signaling pathways including MAPKand JNK that direct cell-cycle progression and differenti-ation, and this conversion of physical signals intobiochemical signals is termed mechanotransduction.

Mechanics of the Natural ECM

In vivo, tissues including bone, arteries, and brain naturallyhave distinct moduli (Black and Hastings, 1998), where themodulus of each is primarily defined by the properties ofthe ECM. Accordingly, ex situ measurements of naturaltissues demonstrate the wide range of stiffnesses ofdifferent tissues in the body. Engler et al. (2006) observedthat the osteoid matrix that surrounds osteoblasts in culturehad a Young’s modulus of ~27 � 10 kPa, much stiffer thanother tissues in the body. In addition, a few years prior,Engler et al. (2004) sectioned arteries ex situ and measuredan intermediate stiffness with a measured Young’s modulusof 5e8 kPa. Finally, Elkin et al. (2007) and Lu et al. (2006)made measurements of native brain tissue, which hada much lower modulus of ~500 Pa. The different moduli ofthese tissues in vivo indicate there may be a significant roleof the stiffness of the matrix in cell fate and cell behavior.

DEVELOPING ARTIFICIAL MATRICESWITH TUNABLE MODULI FOR STEMCELL CULTURE

The physical and chemical properties of the ECM playa key role in stem cell fate; therefore, the field of tissueengineering has the difficult task of creating artificialmatrices that impart the desired signals to the cells in directcontact with those matrices. In addition, since the naturalmicroenvironment is dynamic in nature, it may be neces-sary to create tunable systems that the user is able tomodify, for example to direct progressive processes such ascell fate specification or tissue organization. This sectionfocuses on designing matrices for in vitro stem cell culture,both for maintaining stem cell self-renewal and differenti-ating stem cells into a variety of cell lineages.

Physical Structure of the Matrix

The physical structure, or microarchitecture, of an artificialmatrix must provide appropriate physical signals, present

or allow access to biochemical cues, and permit nutrientand waste exchange. Synthetic matrices should mimicsome aspects of the natural properties of the collagenscaffold and adjacent proteins of the ECM, which consti-tutes a highly hydrated and fibrous network that supportscell attachment, migration, and other functions.

One approach to mimicking the physical structure of theECM is the creation of matrices of nanofibers prepared withelectrospun polymers. In 2003, Yoshimoto et al. grewmesenchymal stem cells (MSCs) on scaffolds created byelectrospinning poly(ε-caprolactone) (PCL). They demon-strated increased osteogenic differentiation on the nano-fiber matrices compared with standard tissue culturesurfaces. In 2006, Nur-E-Kamal et al. cultured mouse ES(mES) cells on a synthetic polyamide matrix whose three-dimensional (3D) nanofibrillar organization resembled theECM/basement membrane and found that this surfacegreatly enhanced proliferation and self-renewal comparedwith propagation on tissue culture surfaces without nano-fibers. This important work indicated that mimicking thenanofiber structure of the ECM can yield enhanced cellbehavior, and subsequent work (discussed on p. 931) hasbuilt upon these efforts to incorporate material designs thatallow tuning of a wide range of mechanical properties.

Another approach to the design of artificial matrices forstem cells is to employ a hydrogel to mimic the physicalproperties of the natural ECM. Hydrogels emulate the highwater content and porous nature of most natural soft tissues.In addition, scaffolds have been designed to enable cells toproteolytically cleave certain domains of the network asthey move through it, allowing for the creation of pores(West and Hubbell, 1999; Schense et al., 2000; Kim et al.,2005; Raeber et al., 2005; Levesque and Shoichet, 2007).Hydrogels can thus provide the diverse physical propertiesof an artificial matrix, while also providing a system thatcan be chemically and mechanically tuned for a desiredapplication.

Choice of Material

For the desired microarchitecture, a variety of both naturaland synthetic polymer chemistries can be employed tocreate the nanofiber or hydrogel systems discussed abovethat offer different material characteristics.

Natural Materials

Naturally occurring polymer components from the ECMcan be isolated and employed as artificial microenviron-ments for stem cell culture. For decades, natural materialseincluding alginate (Barralet et al., 2005), chitosan (Azabet al., 2006), hyaluronic acid (Masters et al., 2004), collagen(Butcher and Nerem, 2004), laminin, fibronectin, and fibrin(Eyrich et al., 2007) e have been used as matrices for

930 VOLUME | 2 Adult and Fetal Stem Cells

a variety of primary cells and cell lines. As they are part ofthe natural ECM, these proteoglycan and protein moleculescontain binding sequences to engage cell surface receptorsand allow for cell attachment and migration. However,disadvantages of using natural, isolated materials includelot-to-lot variability in the signals the matrix presents to thecells, the potential transfer of immunogens to cells, and thepotential for viral or bacterial contamination. Therefore,the primary value of most work using natural materials hasbeen to elucidate the roles of natural ECM molecule(s) oncell fate.

In 2005, Battista et al. employed collagen, fibronectin(FN), and laminin (LM) matrices for the culture of mES-cell-derived embryoid bodies (EBs) in an attempt to directstem cell behavior. They showed that the composition ofthe matrix plays an important role in EB development,where high collagen concentrations inhibited EB differen-tiation, FN constructs stimulated endothelial cell differen-tiation and vascularization, and LM constructs increasedthe percentage of cells that differentiated into beating car-diomyocytes. This work indicates that there is key regula-tory “information,” i.e., ligands, and possibly physical cueswithin these ECM proteins that regulate mES cell fatedecisions.

In addition, several groups have utilized surface arraysfor the high-throughput analysis of how different combi-nations of natural ECM molecules can impact stem cellfate. Flaim et al. (2005) designed a platform to study theeffects of 32 different combinations of collagen I, collagenIII, collagen IV, laminin, and fibronectin on the differenti-ation of mouse ES cells towards an early hepatic fate. Theyidentified ECM combinations that impacted both hepato-cyte function and ES cell differentiation. Soen et al. (2006)printed mixtures of ECM components and signaling factorson a glass surface to generate an array of immobilized“molecular microenvironments” and found the composi-tion of the microenvironment affected the degree ofdifferentiation of primary human neural precursor cells.These studies provide fundamental information thatincreases our understanding of the role of matrix compo-sition on stem cell behavior, which can be harnessed todesign synthetic hydrogels that can offer more precisecontrol over matrix properties and signals presented tocells.

Synthetic Materials

In contrast to natural materials, synthetic polymer hydro-gels offer improved control, repeatability, safety, andscalability. However, it can be challenging to functionalizesynthetic materials with the highly complex bioactivities ofnatural materials. Synthetic materials used commonly fortissue engineering of all cell types include poly(glycolicacid), poly(lactic acid), and their copolymers; polyethylene

glycol (PEG) (Sawhney et al., 1993); polyvinyl (PVA)(Martens and Anseth, 2000); polyNIPAAm (Stile et al.,2004); polyacrylamides; and polyacrylates. Several reviewsdescribe the use of these synthetic polymer matrices for thegrowth of anchorage-dependent, differentiated cells (Lutolfand Hubbell, 2005; Lin and Anseth, 2009; Tibbitt andAnseth, 2009), and these chemistries likewise have poten-tial for use in scaffolds for stem cell culture. Whenselecting polymer chemistry for a particular application,design parameters include the toxicity of the material tothe cells, hydrophilicity, swelling behavior, degradationproperties, interactions the polymer chains have withneighboring cells, biofunctionalization, and crosslinkingproperties.

Hydrogel matrices have been used to support thepotency and differentiation of stem cells. Biodegradablepolymer scaffolds were employed for the differentiation ofhES cells into 3D structures with characteristics of devel-oping neural tissues, cartilage, or liver (Levenberg et al.,2003). These synthetic matrices were superior to theirnatural counterparts in scalability, repeatability, and controlover design parameters. In addition, advanced screeningmethods have been employed to identify hydrogel surfacesfor the self-renewal of pluripotent stem cells (Yang et al.,2009; Derda et al., 2010; Hook et al., 2010). However, thereis still a great deal of work to be done to evaluate varioushydrogels and their influence on stem cell behavior,particularly engineering them with the biochemical andmechanical signals inherent in natural matrix.

Synthetic Materials with Bioactive Ligands

Synthetic hydrogels can be modified with bioactive ligandsto allow cells to attach, proliferate, and/or differentiateupon otherwise inert surfaces as shown schematically inFigure 76.2. Extensive work has been performed to identifybinding sequences in natural ECM molecules (Derda et al.,2007) and then generate short peptides or small recombi-nant proteins encompassing these sequences for incorpo-ration into artificial matrices (Orner et al., 2004; Derdaet al., 2007). The resulting bioactive materials can supportcell receptoreligand adhesion, which enables cells to senseand respond to the stiffness of the matrix. However, manykey parameters must be tuned to control cell and stem cellbehavior, including the ligand identity, presentation, anddensity (Nowakowski et al., 2004; Shin et al., 2004; Yimand Leong, 2005; Beckstead et al., 2006).

Peptides are typically conjugated to hydrogels usingeither primary amines or sulfhydryl groups on the peptidesthemselves. The method of conjugation, as well as spacer-arm length, can be varied to modulate the steric accessi-bility of the peptide sequence to the cell. In addition, it canbe difficult to generate synthetic analogues of the complexmotifs that natural ECM proteins present. In some cases,

FIGURE 76.2 Schematic of a cell

embedded in a 3D synthetic hydrogel.

Integrin receptors bind to pendant cell-

binding domains, and growth factor recep-

tors bind to soluble ligands. Cell-secreted

proteases enzymatically cleave substrates

incorporated into the polymer network,

locally degrading it.

931Chapter | 76 Designing Tunable Artificial Matrices for Stem Cell Culture

the natural conformation of the binding site on the proteincan be more closely approximated by cyclizing shortpeptide sequences that are otherwise linear (Schense et al.,2000). The most common molecules used to mediate cellattachment to synthetic matrices are short peptidesequences (usually between 6 and 15 amino acids) con-taining the consensus sequence arginine-glycine-aspartate(RGD), which is present in several ECM proteins. Manystudies have tethered this peptide to hydrogels anddemonstrated its ability to bind anchorage-dependent cellsthrough a subset of RGD-binding integrins, such as avb3(Massia and Hubbell, 1991; Hubbell, 1995). In anotherapproach, Meng et al. (2010) physisorbed short peptidesequences to cell culture plates to bind specific integrinsidentified on hES cells to create a completely syntheticdefined cell culture system for medium-term self-renewalof hES cells.

Using synthetic matrices functionalized with bioactiveligands, Saha et al. (2008) demonstrated both long-termself-renewal and multipotent differentiation of neural stemcells on interpenetrating networks of polyacrylamide andpolyethylene glycol functionalized with a 15-mer RGDsequence isolated from bone rat sialoprotein (bsp-RGD15).In another example, Li et al. (2006) were able to maintainhESC pluripotency employing synthetic semi-inter-penetrating polymer networks (sIPNs) functionalized withthe same peptide. In addition, Hwang et al. (2006) differ-entiated hES cells into mesenchymal stem cells and thenevaluated their chondrogenic capacity upon encapsulationin poly(ethylene glycol)-diacrylate (PEGDA) hydrogelsfunctionalized with an RGD peptide. This combinationyielded neocartilage within 3 weeks of culture. In another

study, Ferreira et al. (2007) formed a 3D matrix from thenatural polymer dextran, then functionalized the materialwith RGD peptides and microencapsulated VEGF. Theywere able to increase the fraction of cells displayinga vascular marker by 20-fold compared to spontaneouslydifferentiated EBs and propose that this hydrogel enablesthe derivation of vascular cells in large quantities.

Creating Matrices with Tunable Moduli

Mechanical design parameters for artificial matricesinclude elasticity, compressibility, viscoelastic behavior,and tensile strength. Controlling the mechanical propertiesof a material at the cellular level can help elicit a desiredcell response, and, in addition, the bulk mechanical prop-erties of the matrix must be controlled such that the matrixis able to withstand loads that may be involved in down-stream applications.

The mechanical properties of hydrogels can be variedand controlled via chemical synthesis and processing.Hydrogels are composed of long, hydrophilic polymerchains either physically entangled or chemically cross-linked to form a network, and their mechanical propertiescan be chemically altered by controlling crosslinkingdensity (entanglements or chemical crosslinks). For theAAm gels described on page 933, the input crosslinker(bisacrylamide) concentration of the AAm gels was varied,and a linear relationship between input crosslinker densityand gel modulus was found. Based on prior work (seep. 933) (Engler et al., 2006; Saha et al., 2007, 2008; Boonenet al., 2009), tuning the crosslink density of hydrogels mayaid in designing systems to support stem cell self-renewal

932 VOLUME | 2 Adult and Fetal Stem Cells

or differentiation, depending on the desired application. Anincreasing number of studies have illustrated a role ofstiffness in regulating stem cell function in two dimensions,and initial evidence to date indicates that the mechanicalproperties of a material are likely to also influence stem cellbehavior in 3D (Banerjee et al., 2009).

Although a direct correlation with matrix stiffness andbehavior of hES or iPS cells has yet to be demonstrated, Liet al. (2006) proposed that the soft mechanical properties oftheir hydrogels improve the self-renewal of hES cells ontheir defined, synthetic hydrogels. In this work, pNIPAAmhydrogels functionalized with bsp-RGD15 and witha complex shear modulus of ~50e100 Pa (depending on thefrequency of the measurement) and were able to maintainpluripotency in the short-term. Future studies are verylikely to focus on analyzing the effects of stiffness andother mechanical properties on the self-renewal, lineagecommitment, and differentiation of numerous cell types,providing additional key design parameters to control cellfunction for downstream applications.

CHARACTERIZATION OF MATRIXMECHANICS

The mechanical properties of synthetic and naturalmatrices are typically characterized by either atomic forcemicroscopy or rheology, and each is addressed in furtherdetail.

Atomic Force Microscopy

The mechanical stiffness of two-dimensional (2D) hydro-gels can be characterized by force mode atomic forcemicroscopy (AFM). AFMs have been used widely asmicroindenters to probe the physical properties of thematerials (Burnham and Colton, 1989; Tao et al., 1992;Rotsch et al., 1997; Domke and Radmacher, 1998; Dimi-triadis et al., 2002; Irwin et al., 2008). In force mode, theAFM tip is indented into the surface, and the deflection of

FIGURE 76.3 Schematic of the indentation of

a gel sample with a rigid sphere using an AFM.

Adapted from Dimitriadis et al. (2002).

the cantilever is measured as shown in Figure 76.3. Toreduce strain at the point of contact, and ensure uniformcurvature at the point of contact, a bead can be attached tothe cantiliver tip as shown.

The AFM collects data by reflecting a laser off a canti-lever with a known spring constant. The laser is reflectedinto a photodiode (detector) as the tip (on the end ofa cantilever) is indented into the surface and bends inresponse to the force between the tip and the sample. Aconstant force is maintained on the sample by the tip byusing a feedback loop with piezoelectric translators thatadjust the z-axis of the stage.

The elastic response of the underlying material isanalyzed by applying Hertzian mechanical models to theslope of the forceedisplacement curves to estimate theelastic modulus and other material properties or structuralparameters (Dimitriadis et al., 2002). The indentationcurves are then analyzed with a Hertzian mechanics modelwith the following relationship:

F ¼ ð2=PÞ½E=ð1� v2Þ�d2tanðaÞ (1)

where F is the applied force, E is the elastic modulus, n isthe Poisson ratio of the material, d is the indentation depth,and a is the angle of the indenting cone. This modelassumes infinite depth of the sample, and therefore theindentation of the tip into the material must be less than10% of the film thickness or the stiffness of the underlyingsubstrate may be sensed by the AFM tip.

Rheology

The mechanical properties of engineered 3D hydrogelsystems, like natural biological tissues, are viscoelastic andare typically characterized using rheological techniques.The mechanical characteristics of such materials are inter-mediate between an ideal solid and an ideal liquid and aredependent on loading rate and history. Rheometry measuresthe flow and deformation behavior of materials under stress,for example by using rotational parallel-plate devices.

933Chapter | 76 Designing Tunable Artificial Matrices for Stem Cell Culture

Oscillatory strain-controlled parallel-plate rheometersapply a sinusoidal shear strain to a hydrogel and measurethe resulting stress (torque) response. The ratio of theamplitudes and phase difference of the stress and strainwaves provide the storage (elastic), G0, and loss (viscous),G00, moduli. The phase angle,

d ¼ arctanðG00=G0Þ; (2)

indicates the degree to which a material is like an elasticsolid or a viscous liquid, while the complex modulus,

G� ¼���sqrt½ðG0Þ2þ ðG00Þ2�

���; (3)

indicates the overall resistance to shear deformation. Theseproperties are measured over a range of frequencies todetermine their dependence on loading rate. Strain sweepsare performed to define the linear viscoelastic regime andyield point of the material.

Rheology is particularly applicable for the analysis ofenvironmentally responsive and in situ-forming hydrogelsfor tissue engineering and cell biology. Kinetic changes inmechanical properties can be measured as the materialstransition from liquid to solid. Liquids are indicated by low,frequency-dependent elastic moduli and high phase angleswhile solids have high, frequency-independent elasticmoduli and low phase angles.

ROLE OF MATRIX MECHANICS IN STEMCELL BEHAVIOR

The biochemistry, physical architecture, and modulus ofthe microenvironment are all important parameters ininfluencing cell behavior. Cells are traditionally cultured ontissue culture polystyrene (TCPS) and glass, which haveYoung’s moduli of ~108 and ~1010 Pa, respectively: valuesthat are orders of magnitude higher than the moduli of mostnatural tissues, ~102e105 Pa. Given this mismatch inmechanical properties, and given that it has been demon-strated in multiple anchorage-dependent cell types thata material’s modulus impacts cell morphology, cytoskeletalformation, and gene expression, this key parameter must beconsidered in the design of cell culture systems.

Pelham and Wang developed a system to evaluate theeffect of material stiffness on cell behavior (Pelham andWang, 1998). This system was composed of 2D, variablemoduli polyacrylamide (pAAm) gels functionalized withcollagen to allow for cell attachment, and has been sinceemployed by multiple research laboratories to demonstratemodulus dependent behavior of a variety of differentanchorage-dependent cell types. These gels, which greatlycontrast with the current tissue culture polystyrene used forstandard cell culture that is orders of magnitude more rigid(~ GPa), provided moduli that more accurately matchedthose of native tissue. In 1998, Pelham and Wang first

demonstrated that fibroblast and epithelial cell behaviorwere regulated by the mechanical properties of the under-lying synthetic matrix on which the cells were cultured.They found that both focal adhesion and cytoskeletalformation depended on the stiffness of the underlyingpAAm gels. Thomas and Dimilla (2000) cultured humanSNB-19 glioblastoma cells on poly(methylphenyl)siloxane(PDMS) films of variable moduli and showed that theaverage projected cell area decreases by over 60% witha two-orders-of-magnitude increase in compliance. Loet al. (2000) cultured fibroblasts on pAAm gels witha spatial gradient in modulus and demonstrated that thefibroblasts preferentially migrated to the stiffer areas of thegel, a process termed durotaxis, indicating the cells wereable to sample the stiffness of the underlying substrate. Inaddition, by applying mechanical strain to the substratewith a microneedle, they demonstrated that cell movementis also guided by strain in the substrate. In 2004, Engleret al. employed the same pAAm gel system and demon-strated that matrix stiffness affected the cell spreading,actin cytoskeletal formation, and focal adhesion organiza-tion of smooth muscle cells (SMCs), where stiffer gelscause an increase in all three.

Several groups have been able to show that, in addition tovarying a number of properties of differentiated cells, thestiffness of the matrix can regulate the lineage commitmentprocesses of adult stem cells. In 2006, the lab of Engler et al.used the pAAm gel system to test the effect of matrix stiff-ness on the differentiation of adult stem cells. A variation instiffness alone was able to control lineage commitment,where softer matrices resulted in neurogenic commitment,intermediate stiffnesses yielded myogenic commitment, andfinally the stiffest matrices resulted in osteogenic commit-ment. This work suggests that mesenchymal stem cellsdifferentiate according to the stiffness of the environment inwhich they were cultured. Reflecting this observation, Sahaet al. (2008) demonstrated that adult neural stem celldifferentiation also depended on the stiffness of the under-lying matrix. In this work, an interpenetrating polymernetwork (IPN) of AAm and PEG functionalized withbspRGD(15) was employed to demonstrate that softer gels(100e500 Pa) greatly favored differentiation into neurons,whereas harder gels (1,000e10,000 Pa) promoted glialcultures. Recently, Boonen et al. cultured muscle progenitorcells (MPCs) onto pAAm gels of varying stiffness and foundthat proliferation and differentiation were influenced byelasticity (Boonen et al., 2009). An intermediate stiffness of21 kPa was optimal for the proliferation of MPCs, whereonly gels with elasticities greater than 3 kPa led to matura-tion with cross-striations and contractions. Collectively,these studies demonstrate that the stiffness of the matrix isa crucial parameter in designing matrices for stem cells;stiffness collaborates with soluble cues to direct lineagecommitment of the cells.

934 VOLUME | 2 Adult and Fetal Stem Cells

CONCLUSIONS AND FUTUREDIRECTIONS

In engineering matrices for stem cell culture, it is evidentthat ligand identity and presentation, as well as materialarchitecture and mechanical properties, are key designparameters in controlling stem cell fate. Although there havebeen significant advances in the design and synthesis ofartificial ECMs, there is still a great need for more sophis-ticated scaffolds that play an active role in guiding tissueregeneration and functional adaptation of the newly formedtissue. In particular, while it is recognized as a signal to thecells, the modulus of the material is still not often varied andoptimized for the particular application. Future work islikely to increasingly tap into this and other opportunitiesthat materials offer to afford greater control over cell fateand function and thereby enhance the potential of numerousdownstream applications for stem cell engineering.

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