review - circulation researchcircres.ahajournals.org/content/circresaha/109/8/910.full.pdf · this...

Review

This Review is part of a thematic series on Stem Cells, which includes the following articles:

Stem Cells Review Series: An Introduction [Circ Res. 2011;109:907–909]

Biomaterials to Enhance Stem Cell Function in the Heart

Mesenchymal Stem Cells: Biology, Pathophysiology, Translational Findings, and Therapeutic Implications for Cardiac Disease

Role of Cardiac Stem Cells in Cardiac Pathophysiology: A Paradigm Shift in Human Myocardial Biology

Imaging: Guiding the Clinical Translation of Stem Cell Therapy

Side Population Cells

Endothelial Progenitor Cells

Genetic and Pharmacologic Modulation of Stem Cell Function for Cardiac Repair

MicroRNAs and Stem Cells

Epigenetic Modifications of Stem Cells: A Paradigm for the Control of Cardiac Progenitor Cells

Chemical Regulation of Stem Cells

Cell Therapy of Acute Myocardial Infarction and Chronic Myocardial Ischemia: From Experimental Findings to Clinical Trials

Cell Therapy of Heart Failure: From Experimental Findings to Clinical Trials

Cell Therapy of Peripheral Vascular Disease: From Experimental Findings to Clinical Trials

Methodological Issues in Cell Therapy Studies: What Have We Learned From Clinical Studies

Stefanie Dimmeler, Douglas Losordo, Editors

Biomaterials to Enhance Stem Cell Function in the HeartVincent F.M. Segers, Richard T. Lee

Abstract: —Transplantation of stem cells into the heart can improve cardiac function after myocardial infarctionand in chronic heart failure, but the extent of benefit and of reproducibility of this approach are insufficient.Survival of transplanted cells into myocardium is poor, and new strategies are needed to enhance stem celldifferentiation and survival in vivo. In this review, we describe how biomaterials can enhance stem cell functionin the heart. Biomaterials can mimic or include naturally occurring extracellular matrix and also instruct stemcell function in different ways. Biomaterials can promote angiogenesis, enhance engraftment and differentiationof stem cells, and accelerate electromechanical integration of transplanted stem cells. Biomaterials can also beused to deliver proteins, genes, or small RNAs together with stem cells. Furthermore, recent evidence indicatesthat the biophysical environment of stem cells is crucial for their proliferation and differentiation, as well as theirelectromechanical integration. Many approaches in regenerative medicine will likely ultimately requireintegration of molecularly designed biomaterials and stem cell biology to develop stable tissue regeneration. (CircRes. 2011;109:910-922.)

Key Words: myocardial infarction � stem cell therapy � biomaterials � tissue engineering � protein delivery

Original received May 22, 2011; revision received July 19, 2011; accepted July 21, 2011. In June 2011, the average time from submission to firstdecision for all original research papers submitted to Circulation Research was 14.48 days.

From the University of Antwerp (V.F.M.S.), Antwerp, Belgium; Harvard Stem Cell Institute and the Cardiovascular Division (R.T.L.), Department ofMedicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA.

Correspondence to Richard T. Lee, MD, Partners Research Facility, Room 279, 65 Landsdowne St, Cambridge, MA 02139. E-mail [email protected](Circ Res. 2011;109:910-922.)© 2011 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.111.249052

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Healthy myocardial tissue is more than just a collection ofmyocytes, endothelial cells, smooth muscle cells and

fibroblasts. The different cell types comprising adult myocar-dium interact to form a highly organized and dynamiccontractile tissue.1 These cells also interact with the extracel-lular matrix, an essential component of functional myocardi-um. The extracellular matrix contains inotropic stimuli andgrowth signals for adult myocytes, as well as signals formitosis, growth, differentiation, and migration of stem andprogenitor cells.2 When cardiac tissue is damaged by amyocardial infarction (MI), cells die in the infarcted regionand the normal highly organized architecture of extracellularmatrix disappears with the cells.3

Different populations of resident cardiac stem cells havebeen isolated from adult myocardium,4 including humanmyocardium.5 However, cardiac stem cells present in themyocardium seem to be too low in number, have inadequatedifferentiation potential, or receive insufficient instructions tocomplete cardiac regeneration after MI. In the natural healingprocess of MI in mammals, myocytes are replaced primarilyby scar tissue. Generation of a limited number of newmyocytes after MI occurs in the border region of the infarctbut not in the infarct zone itself.6 This zonal selectivity couldbe explained by the proximity of surviving capillaries andpreserved perfusion, but another explanation is that thesurrounding viable myocardium and extracellular matrixcontain crucial signals for recruitment, activation, or matura-tion of stem cells in the heart. This concept could challengeattempts to inject a population of stem cells in the center ofthe infarct zone because these cells may not lead to formation

of mature myocytes, a finding supported by a number ofstudies.4,7–9 A solution to this problem could be deliveryof differentiation and maturation signals at the same time ofstem cell delivery; these signals could be soluble proteins,extracellular matrix, or signals transmitted through cell–cellcontact. Biomaterials are the likely means to deliver thesesignals.

Recently, significant progress has been made in the field ofbiomaterials and the field of cardiac regeneration using stemcells. In this review, we primarily discuss new findings at theintersection of both fields. More specifically, we discussbiomaterials used for stem cell transplantation and methodsfor controlling the biochemical and biophysical microenvi-ronment of transplanted stem cells in the heart. This reviewfocuses on research published in the past 5 years; for reviewsof earlier published work, we refer to references.2,10 We alsodiscuss methods in biomaterials research to enhance engraft-ment and differentiation of stem cells, to increase angiogen-esis, and to enhance electric and mechanical integration ofnewly formed cardiomyocytes. We preferentially cite studiesperformed in the heart but use examples of studies in othertissues as well if promising new biomaterials technologieshave not yet been applied in the heart. For review articlescovering cardiac regeneration using stem cells, we refer toother sources.4,11,12 For review articles covering myocardialtissue engineering, we refer to other sources.13–15 For a reviewon the effects of the extracellular matrix on cell signaling, werefer the readers to other sources.16,17

Cardiac Regeneration Using Stem CellsTo repair the damaged heart, many different stem cells havebeen used in preclinical and clinical studies.4,8,11,12,18–20 Manytypes of cells injected in the heart have been shown toimprove cardiac function but at the same time, in almost allstudies, injected cells have a low survival rate after transplan-tation.4 Plausible explanations for this improved cardiacfunction despite a lack of survival and transdifferentiationinto myocytes are secretion of paracrine signals that act onsurviving myocardium4,21 or stimulation of endogenous re-pair.22 Here, we assume that the ultimate goal of stem celltransplantation is formation of new and functional myocardialtissue, and thus we focus on enhancement of the function ofstem cells that have high potential for differentiation intocardiomyocytes. However, many studies on the effects ofbiomaterials on stem cells have used bone marrow-derivedmesenchymal stem cells (MSCs) because of their availabilityand relatively easy culture. Although the differentiationpotential of MSCs into cardiomyocytes is limited,4 in someplaces we cite studies describing promising new biomaterialsusing MSCs if studies have not yet been performed withpluripotent stem cells. For the same reason, in some placeswe cite studies using bone marrow-derived hematopoieticstem cells or endothelial progenitor cells (EPCs). For adetailed description of stem cells used for cardiac regenera-tion, we refer to other reviews.4,11,12

Cells with stem cell properties have been isolated from theadult myocardium, including human.5,9,23–25 At least sometypes of cardiac stem cells can form cells with properties ofall major cardiac lineages, including myocytes, endothelial

Non-standard Abbreviations and Acronyms

BMP bone morphogenetic protein

CSC cardiac stem cell

EF ejection fraction

EPC endothelial progenitor cell

ESC embryonic stem cell

FGF-10 fibroblast growth factor-10

FS fractional shortening

IGF-1 insulin-like growth factor-1

iPS induced pluripotent stem cell

MI myocardial infarction

MNC mononuclear cell

MSC mesenchymal stem cell

PCL poly�-caprolactone

PEG polyethylene glycol

PEU polyether urethane

PGA polyglycolic acid

PLCL polylactide-co-�-caprolactone

PLGA polylactic-coglycolyc acid

PLLA polyL-lactic acid

PNIPAAM polyN-isopropylacrylamide

RGD arginine–glycine–aspartic acid

SDF-1 stromal cell-derived factor-1

Segers and Lee Biomaterials to Enhance Stem Cell Function 911



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cells, and fibroblasts, both in vitro and in vivo.5,23 Potent-ial cells for transplantation include induced pluripotent stemcells (iPS),26–29 which most commonly are skin fibroblaststhat have been transformed into pluripotent cells. The iPScells are potentially one step closer to the “ideal” stem cell forcardiac regeneration because they have the differentiationpotential of embryonic stem cells (ESCs),27 but potentiallywith less risk of teratogenicity30 and fewer ethical concerns.31

However, iPS cells could be biased toward differentiationinto certain lineages because of epigenetic memory, in whichiPS cells retain information of the parental source andremnants of the reprogramming process.32 The ideal stem cellshould be easy to isolate, should have a high survival rateafter delivery, and should form contractile and nonarrhyth-mogenic cardiac tissue. Furthermore, cell preparation meth-ods must be low-cost, robust, scalable, and use chemicallydefined materials.33 Of note, a recent study indicated thatinduction of complete pluripotency for iPS cells might not benecessary to generate beating cardiomyocytes.34–36

In this review, we focus on delivery of multipotent andpluripotent stem cells and not on delivery of their differenti-ated progeny. Major drawbacks of the injection of pluripotentstem cells are the potential for teratogenicity and for misdi-rected differentiation into unwanted lineages.37,38 Onemethod to overcome these drawbacks is differentiation ofESCs into cardiomyocytes in vitro before transplantation.Differentiation protocols for ESCs have been optimized andshow robust differentiation into myocytes in vitro.39,40 ESC-derived cardiomyocytes have been incorporated in differentbiomaterials for transplantation in the heart.41–43

Even if the ideal cardiac stem cell exists or could bedesigned, infarcted myocardium may not provide the tempo-ral and spatial signals present in viable myocardium that arenecessary for adequate survival, proliferation, differentiation,and functional incorporation into organized myocardial tis-sue. Engineered biomaterials likely will be necessary todeliver these signals to transplanted stem cells.

Biomaterials Used for Stem CellTransplantation in the Heart

Many different definitions exist for biomaterials, with onebroad definition being materials used as a medical device forimplantation.10 Historically, most research on biomaterialsfocused on orthopedics and development of prosthetics. Withmore sophisticated medical devices, the emphasis on bioma-terials moved toward biocompatibility. More recently, novelmolecularly designed biomaterials have been developed thatcan deliver growth factors and control the environment oftransplanted cells.

Biomaterials injected on their own—without inclusion ofcells—can decrease remodeling after MI by increasing thick-ness of the infarct, resulting in decreased wall stress onsurviving myocardium.15 This effect is more pronouncedwhen the passive mechanical properties of the biomaterial aresimilar to those of healthy myocardium.15 For instance, aninjectable alginate hydrogel has been developed for intramyo-cardial44 or intracoronary45 injection after MI that increasesthe wall thickness in the infarcted area. Injection of thisalginate hydrogel results in a larger myocardial mass and a

decreased end-diastolic volume.44,45 However, the effects ofbiomaterials without stem cells might be temporary; a recentstudy in which a polyethylene glycol hydrogel was injected ininfarcted rat hearts showed a decrease in end-diastolic vol-ume 4 weeks after MI,46 but benefits disappeared after 3months. This study suggests that the positive effects on wallstress after injection of biomaterials without cells mightdisappear when the biomaterials are degraded.

Biomaterials used for cardiac regeneration ideally willhave properties of myocardial extracellular matrix, includingsimilar passive mechanical properties, signals for cellularattachment, and signals for proliferation and differentiation.In addition, biomaterials that are no longer needed willpreferably degrade without toxic metabolites and with acontrolled degradation rate. Biomaterials should also result inlimited foreign body reaction and limited inflammatory re-sponse. Foreign body reaction with giant cells can occur withinert biomaterials like polyethylene glycol (PEG).46 Myocar-dial biomaterials should last long enough to guide integrationof transplanted cells, but not long enough to interfere with theessential physiological coupling between differentiatedcells.10 Furthermore, viscosity of injectable biomaterials pref-erably will be low enough to allow injection through long andnarrow catheters, but at the same time solidification should befast enough to prevent washout of the injected cells. Differentinjectable biomaterials have been used to deliver stem cells tothe heart (Tables 1 and 2), including matrigel,47 collagen,48–51

fibrin,50,52 Polylactic-coglycolic acid (PLGA),53 self-assembling peptides,54–57 and alginate.44,45 In this section, weprovide an overview of biomaterials most commonly used forstem cell delivery in preclinical studies.

MatrigelMatrigel is a solubilized protein preparation extracted fromEngelbreth-Holm-Swarm mouse sarcoma, a tumor rich inbasement membrane proteins.58 The major component ofmatrigel is laminin, followed by collagen IV, heparan sulfateproteoglycan, and entactin.58 Matrigel is a liquid at lowtemperatures but forms a hydrogel within minutes at 37°C.Therefore, matrigel can be used as an injectable hydrogel fordelivery of stem cells to cardiac tissue. It has been shown thatmatrigel increases the retention of ESCs when injected afterMI, and a combination of matrigel with ESCs improvescardiac function after MI.47 Because matrigel is a materialsecreted by living cells, it contains many growth factors thatvary from batch to batch, and this could have a variableimpact on growth and differentiation of stem cells. Further-more, matrigel is derived from a mouse tumor and thereforeis not appropriate for clinical translation.

CollagenCollagen is a ubiquitous extracellular matrix protein thatprovides tensile strength to tissues. Turnover of collagen is animportant factor in the formation of scar tissue in the infarctzone and in the remodeling of cardiac tissue in the remotezone after MI. Collagen also can be used as a substrate forthree-dimensional in vitro cell culture and for in vivo tissueengineering applications. It is an inexpensive, nontoxic,nonimmunogenic, and biodegradable compound. Because of

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its viscosity, collagen prevents redistribution of MSCs in-jected into the infarcted myocardium to remote organs.48,50

Collagen gels have been used as a supporting matrix intissue-engineered constructs of EPCs.49 These constructs thenhave been implanted epicardially in rats after MI and havebeen shown to improve cardiac function by increasing vas-cular density.49 Using a percutaneous approach, collagen gelsalso have been used to deliver MSCs or viral particles into thepericardial space in large animals.59

FibrinFibrin is a fibrous protein that plays an essential role in bloodclotting. A fibrin matrix is formed within seconds when

fibrinogen is mixed with thrombin, a method on whichcommercially available fibrin glue is based.60 A major ad-vantage of fibrin as a biomaterial is its availability of a Foodand Drug Administration-approved product. Similar to colla-gen, two main strategies have been pursued to deliver stemcells to the heart using fibrin: construction of a fibrin patchfor epicardial application52 or the use of fibrin as an injectablebiomatrix together with stem cells.50 A third strategy isepicardial application of fibrin glue immediately after and atthe site of injection.61 When cells are injected through theepicardium during surgery, application of fibrin glue in-creases retention of injected stem cells.61 Fibrin also has beenused to deliver proteins, plasmids, and viral vectors for

Table 1. Biomaterials Used to Enhance Stem Cell Function in Preclinical Models of Myocardial Infarction

Biomaterial Stem Cells Factor Animal Functional Benefit Mechanism Reference

Matrigel ES cells NA Mouse FS�15.2% NA 47

Collagen MSCs NA Rat NA Retention 48

MSCs NA Rat �dP/dT�1800 mm Hg/s Retention 50

MSCs NA Rat FS�6% NA 51

EPCs SDF-1 Rat EF�28% Angiogenesis 49

ESC-derived CM NA Rat NA Engraftment 42

Fibrin MSCs NA Rat �dP/dT�800 mm Hg/s Retention 50

MSCs NA Pig NA Angiogenesis 52

Self-assembling peptides MNCs NA Pig EF�24% Retention Angiogenesis 54

ES cells FGF-10 Mouse NA Differentiation 110

CSCs IGF-1 Rat EF�8.4% Maturation 74

CSCs NA Mouse FS�9.7% Angiogenesis 119

MSCs RGD Rat EF�24.4% Retention Differentiation 68

PLCL MSCs NA Rat EF�23% Infarct size 120

CSC indicates cardiac stem cell; dP/dT, rate of rise of left ventricular pressure; EF, ejection fraction; EPC, endothelial progenitor cell; ES cells, embryonic stem cells;FGF-10, fibroblast growth factor-10; FS, fractional shortening; IGF-1, insulin-like growth factor-1; MNC, mononuclear cell; MSC, mesenchymal stem cell; NA, notapplicable; PLCL, polylactide-co-�-caprolactone; RGD, arginine–glycine–aspartic acid; SDF-1, stromal cell-derived factor-1.

Table 2. Properties of Selected Biomaterials for Tissue Regeneration

DegradationProducts Degradation Time

InflammatoryResponse/Toxicity

Cell Viability andProliferation

Cell DifferentiationMorphology

Natural Biomaterials

Matrigel Amino acids58 Low N of neutrophils121 1 Retention47 1 Differentiation58

Collagen Amino acids 1 Retention48,50

1 Survival122

Fibrin Amino acids 1 Retention50 1 Vasculature 123

Alginate Saccharides44 4–6 wk44 1 Viability124 1 Attachment124

Synthetic Biomaterials

Self-assembling peptides Amino acids 2 wk57 1 Retention54 1 Differentiationwith IGF-174

PGA Glycolic acid125 3 wk125 Dose dependent toxicity126 1 attachment 127

PLLA Lactic acid126 Depending on MW128 Dose dependent toxicity126

PLGA Lactic acid,53 glycolic acid �3 wk125

PEG Nondegradable46 �13 wk46 Macrophages,46 foreign body46 1 Cell aggregation129

PLCL Lactic acid,120 caprolactone 1 Retention130 1 Differentiation130

PCL Caprolactone131 2 wk131 Dose dependent toxicity126 1 Attachment132

IGF indicate insulin-like growth factor; MW, molecular weight; PCL, poly-�-caprolactone; PEG, polyethylene glycol; PGA, polyglycolic acid; PLCL, polylactide-co-�-caprolactone; PLGA, polylactic-coglycolyc acid; PLLA, poly-L-lactic acid.




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applications in many different tissues and provides sustainedrelease for up to 2 weeks.60 When used to deliver MSCs to theheart after MI, fibrin increases retention of MSCs50 andincreases angiogenesis.52

Decellularized Extracellular MatrixDecellularized porcine heart valves are now commonly usedin clinical practice, but decellularized whole organs or tissueconstructs are a relatively new research area. Complete heartscan be decellularized by perfusion with detergents and usedas a scaffold for implantation of myocytes and endothelialcells.62 These recellularized constructs show contractile func-tion, although limited.62 Ultimately, this strategy could lead totransplantation of in vitro engineered hearts using decellular-ized exogenous matrix and autologous stem cells. An alter-native approach is lyophilization and solubilization of decel-lularized extracellular matrix,63,64 a method that results in asoluble and injectable biomaterial similar to native extracel-lular matrix. This myocardial matrix could be used to deliverstem cells and it is possible that it contains cues for cardiaclineage commitment.

Polylactic-coglycolic AcidPLGA is a copolymer used in manufacturing of biodegrad-able sutures and has been approved by the Food and DrugAdministration for drug delivery approaches.53 This polymeris degraded to nontoxic lactic and glycolic acid moieties, andthe degradation rate can be modified by adjusting the ratio oflactic to glycolic acid.53 PLGA is used for the manufacturingof microparticles for drug delivery. Commonly used methodsfor PLGA microparticle formation are single-emulsion sol-vent evaporation or double-emulsion solvent evaporation.The single-emulsion method uses an emulsification of oil inwater, whereas the double-emulsion process involves emul-sification of water in oil in water in which small waterdroplets are incorporated into the organic phase duringmanufacturing.53 Oil-in-water emulsions allow for delivery ofhydrophobic drugs,65 whereas water-in-oil-in-water emul-sions allow for delivery of hydrophilic molecules, includingDNA and proteins.53,65 However, polymerization of PLGA isa harsh chemical process that can lead to denaturation ofproteins and that restricts seeding of cells after polymeriza-tion has been completed.66 Therefore, PLGA has been usedprimarily to generate tissue engineered scaffolds. A majoradvantage of PLGA scaffolds is that they can be used formany different applications; an example is printing of apattern of 20-�m-wide fibronectin strips on PLGA sheets.67

This pattern stimulates a more elongated morphology ofMSCs instead of their classical flattened growth and leads tomyogenic differentiation.67 Thus, control of cell shape withbiomaterials could be a novel method to guide differentiationof stem cells into myocytes.

Besides PLGA, other synthetic biomaterials like PEG46 orpoly-N-isopropylacrylamide (PNIPAAM)63 have been shownto be injectable in the myocardium. However, delivery ofstem cells in the myocardium has not been studied yet.Furthermore, many synthetic materials like poly-L-lactic acid(PLLA)41 have been used to culture stem cells or cardiomyo-cytes in vitro but have not been used in vivo yet.

Self-assembling PeptidesSelf-assembling peptides are small peptides typically consist-ing of 8 to 16 amino acids in a sequence with alternatinghydrophobic and hydrophilic residues.56 They are soluble inwater and at low pH but form a stable hydrogel of nanofiberswithin minutes at physiological pH and salt concentrations.56

This rapid responsiveness to physiological conditions makesthem an attractive biomaterial for injection into tissues. Othermajor advantages of self-assembling peptides are that theycan be manufactured easily by standard chemical peptidesynthesis and that they are degraded into natural amino acidsover a time period of weeks.57 Self-assembling peptidenanofibers can be used for three-dimensional cell culture andare nontoxic.56

Self-assembling peptide nanofibers improve retention ofbone marrow mononuclear cells 4 weeks after transplantationwhen injected intramyocardially in pigs with MI.54 Further-more, peptide nanofibers also stimulate differentiation ofbone marrow cells into vascular endothelial and smoothmuscle cells and can improve both systolic and diastolicfunction.54 As described, self-assembling peptides can beengineered for protein delivery56 and adhesion signals can beincorporated into the peptide sequence. For instance, it hasbeen reported that incorporation of the common adhesionmotif RGD (arginine–glycine–aspartic acid) in the sequenceof self-assembling peptides improves survival of MSCs afterinjection in infarcted rat hearts.68 This study also suggestedthat the RGD motif increased differentiation of MSCs incardiomyocytes.68 Furthermore, incorporation of cell-specific adhesion signals in hydrogels also can directdifferentiation into certain lineages. For example, incorpo-ration of the neurite-promoting laminin epitope peptidesequence of isoleucine-lysine-valine-alanine-valine in ahydrogel of self-assembling peptide nanofibers guidesdifferentiation of neural progenitors selectively towardneurons instead of astrocytes.69,70

Controlling the BiochemicalMicroenvironment of Stem Cells

Protein DeliveryBiomaterials can be used to deliver small and large mole-cules, including proteins, to tissues (Figure).56 Many proteinsdiffuse rapidly from the injection site or are degraded rapidlyby proteases present in inflammatory environments likeinfarcted tissue. Furthermore, control of temporal and spatialdistribution of delivered proteins may be critical for effectiveresponses by target cells.10 For instance, spatial distribution isimportant for proteins with chemotactic properties; thesechemokines need to be delivered in such a way that achemotactic gradient can be formed.56 When a gradient ofa chemokine is too steep, cells can be repelled instead ofattracted, which is a phenomenon called fugetaxis.71 Tempo-ral distribution is particularly important for differentiationfactors; expression of a specific differentiation factor at theright time and place is crucial for normal cardiogenesis.1,72

During embryonic development, stem cells are highly sensi-tive to a fine-tuned temporal orchestration of Wnt, fibroblastgrowth factor (FGF), BMP, Hedgehog, and Notch signaling




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pathways.73 A given signal might promote differentiation at acertain stage during development but might inhibit differen-tiation at another stage.73 Considering the number of proteinsparticipating and the delicate temporal and spatial orchestra-tion of these signals, controlled delivery of all signalingmolecules that promote cardiac differentiation with a singleinjection of a biomaterial is a daunting challenge. Identifica-tion of the key signaling factors will be crucial to define alimited number of proteins that can be engineered for deliveryapproaches with stem cells.

Different strategies have been developed for local deliveryof proteins and to prevent rapid diffusion from the site ofinjection. Timed release systems generally contain an aque-ous phase with soluble protein embedded in a degradablephase, which is most commonly an organic polymer. Anexample is PLGA, in which small aqueous particles contain-ing the protein are released over time by hydrolysis of thesurrounding PLGA solid phase.53 Degradation properties andthus protein release kinetics can be altered by changing theratio of lactic to glycolic residues. A drawback of thistechnology is that a number of proteins are degraded by thechemical process during polymerization.53 An alternativeapproach is delivery of proteins by tethering them to hydro-gels.56 For example, hydrogels consisting of self-assemblingpeptide nanofibers can be used for delivery of proteins likeinsulin-like growth factor-155,74 or stromal cell-derivedfactor-1 (SDF-1).57,75 Biotinylated insulin-like growthfactor-1 has been tethered to biotinylated self-assemblingpeptides using streptavidin as a linker,55 which allows deliv-ery of insulin-like growth factor-1 to the heart for more than1 month.55 It has been shown that delivery of insulin-likegrowth factor-1 in this manner, together with cardiac stemcells, leads to a greater improvement of cardiac function afterMI compared to cells alone and to a development of moremature myocytes.74 Instead of using a biotin–streptavidinlinker, another approach is to make fusion proteins of theprotein to be delivered and the 16-amino acid sequence ofself-assembling peptides.57,75 For instance, a recombinantfusion protein of SDF-1 and the sequence of self-assemblingpeptides has been stably incorporated in a hydrogel of

self-assembling peptide nanofibers.57,75 This approach led toincreased homing of EPCs to the heart, increased angiogen-esis, and improved cardiac function.75 Another system thathas been designed for delivery of paracrine factors usesheparin-presenting nanofibers.76 This system can be used todeliver heparin-binding proteins like vascular endothelialgrowth factor or basic fibroblast growth factor.76

Gene DeliveryLocal delivery of plasmids or viruses in myocardium couldpotentially enhance stem cell therapy.77 Gene therapy cur-rently lacks precise quantitative control of expression, butsome applications may tolerate broad ranges of overexpres-sion. Biomaterials can sustain delivery, protect plasmid DNAfrom degradation, and increase transfection efficiency.78 Dif-ferent biomaterials have been used to deliver plasmids andviral vectors,79 including fibrin,60 water-soluble lipopoly-mers,80 or RGD-conjugated bioreducible polymers. Be-cause transplanted cells can be transfected more efficientlyin vitro before injection, the combination of DNA deliveryby biomaterials with stem cell delivery has little promise;a potential application could be delivery of proangiogenicplasmids81 with stem cells to increase survival and suc-cessful engraftment.

MicroRNA DeliveryMicroRNAs (miRNAs) are emerging as critical modulatorsof cardiovascular development and disease.82,83 MiRNAs aresingle-stranded RNAs of 22-nucleotides that inhibit the ex-pression of specific mRNA targets through base pairingbetween the miRNA and sequences located in the untrans-lated regions of the target mRNAs. It is estimated that thehuman genome encodes up to 1000 miRNAs.82 An importantrole of miRNAs seems to be the “fine-tuning” of geneexpression during tissue development and tissue homeosta-sis.82 Targeting miRNAs raises exciting new possibilities toenhance stem cell function. For instance, formation of newblood vessels is regulated by miRNAs,83 both by proangio-genic and antiangiogenic miRNAs. Delivery of antagonists ofantiangiogenic miRNAs could be a novel angiogenic therapy

Figure. Control of the biochemical andbiophysical microenvironment of trans-planted stem cells. Biomaterials can beused to deliver proteins (A), genes (B), ornucleotides interfering with microRNAs(miRNAs; C). D, Nanoparticles containingdrugs or proteins can be linked to trans-planted stem cells and provide a localdepot for long-term release of growth anddifferentiation factors. E, Mechanical stiff-ness of biomaterials can guide differentia-tion of stem cells toward certain lineages.F, Small patterns of the biomaterial,called nanotopography, have an influenceon growth and differentiation of stemcells. G, Cyclic stretch is a typical featureof the cardiovascular system and hasbeen shown to guide stem cell differentia-tion toward myocytes or smooth musclecells. H, Biomaterials can alter geneexpression patterns by controlling cellularshapes. (Illustration credit: Cosmocyte/Ben Smith.)




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in the cardiovascular system. An example of a highly ex-pressed antiangiogenic miRNA in endothelial cells is miR-92a.84 Administration of an antagomir against miR-92a in-creased angiogenesis in a model of hind limb ischemia.84 Thisantagomir potentially could be used to enhance stem cellfunction after MI by decreasing tissue ischemia.

For experimental RNA delivery, two different approachesare currently pursued.85 One approach involves delivery ofsynthetically produced oligonucleotides into the desired cellsby way of targeting agents, chemical modifications, or directadministration in the organ. Another approach is delivery ofgenetic material into the nucleus using viral vectors tooverexpress small RNA precursors using the cell’s genetranscription machinery.85 Viral vectors have some advan-tages over oligonucleotides: stronger levels of “knockdown”of gene expression, the treatment needs to be performed onlyonce because levels are maintained by transcription, and thedose remains constant even in dividing cells.85 As with genetherapy approaches, however, expression levels are difficultto control with viral vectors. Therefore, most therapeuticprograms targeting miRNAs are working on synthetic oligo-nucleotides, which may have more predictable pharmacoki-netics. However, getting synthetic RNAs into the cytoplasmis challenging because of their size and charge. Furthermore,unmodified RNA is cleaved rapidly by nucleases in theplasma, which raises the need for modifications.

Similarly to protein therapy and gene therapy, the pharma-cokinetics of miRNA-based therapies remain a hurdle, be-cause targeting of expressed mRNAs raises possibilities foreffects in other organs.82 This might necessitate the use oflocal delivery methods developed for protein or gene deliveryor the use of targeted delivery strategies. A recent example ofa biomaterial used for targeted delivery of small RNAs is theuse of nanoparticles consisting of a cyclodextrin-based poly-mer, PEG as a stabilizer, and a ligand to a transferrin receptorfound on the surface of the cancer cells.86 This strategy iscurrently used in a clinical trial targeting melanoma cells.86 Inanother study, PCL nanofibers have been used to create asustained release vehicle delivering small RNAs for at least28 days.87

Cell-Conjugated NanoparticlesA recent study described a promising new strategy to deliverdrugs or proteins together with transplanted cells.88 Syntheticdrug carrier nanoparticles were coupled to the surface ofT-lymphocytes using free thiols on cell surface proteins.88

Liposome-like synthetic nanoparticles of 100 to 300 nm indiameter with a drug-loaded core and phospholipid surfacelayer were covalently linked to free surface thiols usingthiol-reactive maleimide head groups incorporated in the lipidbilayer of the nanoparticles. Particle conjugation wasachieved by a two-step process in which donor cells were firstincubated with nanoparticles to permit maleimide-thiol cou-pling, followed by PEGylation to quench residual reactivegroups of the particles.88 Conjugation of these drug-loadednanoparticles to the surface of T-cells did not cause toxicityto the cells, did not interfere with cell function, and led tosustained drug release in proximity of the donor cells.88

Although this approach has not yet been used in the heart, this

novel strategy could be an efficient tool for delivery of smallmolecule drugs, siRNAs, and proteins together with trans-planted cells. The strategy has several advantages, such asactive compounds are released over time and in close prox-imity of the transplanted cells, high local concentrations ofthe compounds, and less off-site effects.

Controlling the Physical Microenvironment ofStem Cells

Most research on stem cell differentiation has been dedicatedto identification and modification of soluble biochemicalfactors in the microenvironment. These biochemical factorsinclude proteins that bind to cell surface receptors and act asgrowth and differentiation signals. However, the physicalmicroenvironment of stem cells also plays an important rolefor differentiation, growth, and integration of stem cells.89

Stem cells, including cardiac stem cells, have been shown tocluster in stem cell niches at specific anatomic locations andwith a specific physical microenvironment.24,25 Surprisinglylittle is known about which physical characteristics of theextracellular matrix in these niches are important for stem cellproliferation and differentiation. The characteristics of thephysical microenvironment that have been shown to modu-late stem cell fate in different tissues include extracellularmatrix stiffness,90 interactions with nanoscale features,91

cyclic stretching,92,93 and cell shape67,94 (Figure). Impor-tantly, most studies examining the effects of the physicalmicroenvironment on stem cell fate have been performed onadult stem cells such as MSCs,89 which are not pluripotentand have less cardiomyogenic potential compared to ESCs.

Extracellular Matrix StiffnessExtracellular matrix stiffness and elasticity can be importantfactors determining stem cell fate. For example, the physicaleffects of matrix elasticity are important for expansion andproliferation of hematopoietic stem cells.95 Culturing primi-tive hemopoietic cells on tropoelastin, which is an elasticbiomaterial, leads to a two-fold to three-fold expansion ofundifferentiated cells in vitro compared to culture on non-elastic biomaterials.95 Furthermore, differentiation of MSCsinto different lineages is sensitive to the elasticity or stiffnessof the extracellular matrix.90,96 Soft matrices mimicking braintissue are neurogenic, stiffer matrices mimicking muscletissue are myogenic, and rigid matrices mimicking bone areosteogenic.90 When MSCs are cultured in vitro, reprogram-ming is possible with addition of soluble factors during thefirst week of culture, but after several weeks MSCs commit tothe lineage specified by matrix elasticity and not by solublefactors present.90 MSCs sense matrix elasticity by pullingagainst the matrix with nonmuscle myosin II isoforms that arepart of the cellular motors.90 This information affects geneexpression by signaling through cellular mechano-transducers.90 For the purpose of cardiac regeneration afterMI, biomaterials could be designed with the optimal stiffnessfor a particular stem cell, for instance, by varying the numberof cross-links between polymers.

NanotopographyCells respond to topographical properties of the extracellularmatrix on a nanometer scale by adhesion molecules on their




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cell surface. Nanotopography can be important in the designof new tissue engineering materials because nanoscale di-mensions of the surface are capable of modulating cellularresponses.97 Nanotopography can influence cell morphology,adhesion, motility, proliferation, protein expression, and generegulation.97 Nanotopography also contains important cuesfor the differentiation of stem cells. It has been shown thatboth the cell bodies and nuclei of MSCs become elongatedwhen cultured on nanopatterns with gratings of 350 nmwidth.91 Muscular and neuronal gene markers were signifi-cantly upregulated in MSCs cultured on these nanopatterns.91

These results indicate that differences in molecular confor-mation, surface topography or roughness,98 fiber diameter, orother nanoscopic modifications of biomaterials can guidedifferentiation of stem cells into myocytes. Furthermore,electric properties like action potential, conduction velocity,and the expression of a cell–cell coupling protein are sensi-tive to differences in the nanoscale features of the surround-ing extracellular matrix.99 For example, tissue constructs ofneonatal rat ventricular myocytes on a hydrogel guided bynanoscale mechanical cues display anisotropic action poten-tial propagation and contractility characteristic of the nativetissue.99 Some features like surface topography or roughnessare more easily modified in solid compared to liquid bioma-terials, whereas other features like fiber diameter are moreeasily modified in hydrogels.

Cell shape can be modified by printing patterns of adhesionsignals like fibronectin on biomaterials in the micrometerrange. Printing of a pattern of 20-�m-wide fibronectin stripson PLGA sheets stimulates a more elongated adhesion ofMSCs and leads to myogenic differentiation.67 Shape-dependent control of lineage commitment has been shown tobe mediated by RhoA activity via ROCK-mediated cytoskel-etal tension.94 Gene expression in cells also can be modifiedby cell shape distortion with micropatterns on biomaterials.Manipulating cellular shape may be a promising method toguide differentiation of stem cells into myocytes.

Cyclic StrainCells in the myocardium and arteries are subjected to constantcyclic strain, and cyclic stretch might guide differentiation ofstem cells into myocytes or smooth muscle cells. MSCscommit to the smooth muscle lineage when they are subjectedto cyclic strain in one direction (uniaxial), but not when theyare subjected to strain in two directions (equiaxial).92 Uniax-ial strain better-mimics circumferential strain experienced bysmooth muscle cells in arteries.93 Biomaterials could bedesigned in such a manner that they are stiffer in onedirection, allowing control of the direction of strain transmit-ted to the transplanted cells and thus guiding differentiation.

ESCs are also responsive to cyclic stress; cyclic stresstransduced on ESCs via focally attached magnetic beadsinduces spreading and differentiation in mouse ESCs, but notin more differentiated cells.100 This response of ESCs isdictated by the cell softness, suggesting that less differenti-ated (softer) ESCs are more responsive to focal cyclic stress.The finding that softness of mouse ESCs makes themsensitive to local cyclic stress suggests that small local forces

might play important roles in embryogenesis and in stem celldifferentiation in adult animals.

Biomaterials to Enhance Stem CellEngraftment and Angiogenesis in the Heart

Most stem cells are small cells, and when they are injected inthe myocardium a substantial proportion leaves the heartwithin minutes.101,102 Because many injectable biomaterialsincrease viscosity of the injected cell suspension, they canalso increase retention of cells. This is an easy but effectiveway by which biomaterials can increase efficacy of stem celltransplantation. Some biomaterials, like self-assembling pep-tides or matrigel, are liquid in solution but rapidly form ahydrogel when injected in physiological salt concentrationsor temperatures.56 This adaptive property increases retentionof injected cells without dramatically increasing viscosity ofthe injectate.

Long-term survival of stem cells is another important goalof stem cell therapy but is more challenging to improve thanearly cell retention. Infarcted myocardium is an ischemic andinflammatory environment hostile to most cells. Biomaterialswith anti-inflammatory properties can enhance survival ofcells.103 Some biomaterials enhance angiogenesis104 and de-crease ischemia in the time frame of a few weeks. Becauseformation of functional vessels tends to be a slow process,successful stimulation of angiogenesis might be a viablestrategy to increase stem cell engraftment.

In the adult myocardium, endothelial cells outnumbermyocytes by 3:1105 and are in close proximity of everymyocyte. Endothelial cells not only form blood vessels andprovide oxygen and nutrients to the myocytes but also exerttrophic and inotropic effects on myocytes.105 Despite the factthat endothelial cells are a necessary part of myocardium,clinical trials with angiogenic proteins like vascular endothe-lial growth factor and FGF-2 have not resulted in an angio-genic therapy that improves cardiac function.106 Biomaterialshave the potential to promote angiogenesis and to supportother types of transplanted cells.

Ideally, transplanted stem or progenitor cells will form newand functional myocytes. Thus far, in at least some reportsusing bone marrow stem cells, the primary mechanism mightbe an increase in capillary density.4,107,108 An explanation forthis improved cardiac function in these trials could beparacrine signaling from newly formed capillaries. When thetransplanted stem cell type mainly differentiates into endo-thelial cells, additional angiogenic stimuli might be lessuseful. However, when the transplanted cell type mainlydifferentiates into myocytes, biomaterials could play a sup-portive role by delivering angiogenic factors and stimulatingangiogenesis. For instance, self-assembling peptide nanofi-bers have been used to deliver SDF-1, a chemotactic proteinfor EPCs.75 Delivery of SDF-1 not only increased capillarydensity but also improved cardiac function after MI.75 Thistechnology could be delivered to infarcted myocardiumtogether with stem cells with myogenic potential, resulting innew myocyte formation supported by angiogenesis providedby self-assembling peptide nanofibers.




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Enhancing DifferentiationDuring development and in the adult organism, the naturalextracellular matrix contains cues for self-renewal and differ-entiation of stem cells.1 Only recently, however, biotechno-logical applications have been developed using these cues forcell proliferation and differentiation. For example, a systemhas been described109 for culturing iPS cells on recombinanthuman laminin-511, a component of the natural niche ofESCs. When small clumps of human ESCs or iPS cells areplated on laminin-511, cells spread out in a monolayer andmaintain cellular homogeneity. This homogeneous mono-layer of iPS cells provides controllable conditions for studyand design of differentiation methods.109

During embryonic development, transitioning from prolif-erative progenitors to differentiated cardiomyocytes requiresboth downregulation of proliferation signals and upregulationof differentiation signals.72 Delivery of differentiation factorstogether with ESCs or iPS cells is a promising but challeng-ing approach because the specific temporal and spatialwindow in which these cells are responsive to variousdifferentiation factors. Chan et al110 recently showed thatFGF-10 doubles the amount of cardiomyocytes formed invitro. Furthermore, when FGF-10 was delivered with self-assembling peptide nanofibers together with ESCs in theinfarct zone in mice, the number of troponin-positive newlyformed cells doubled compared to the group without FGF-10.110 Although these results are promising, this research areais in its infancy and more knowledge on differentiationfactors is needed before a significant number of maturemyocytes are formed with this approach.

Enhancing Electric and MechanicalIntegration in the Heart

During development, rounded cardiomyocyte precursors be-come elongated and align themselves end-to-end in a patternof parallel organized bundles. Electric conduction parallel tothe bundles of myocytes is three-times faster than conductionperpendicular to the bundles.111 MI followed by scar forma-tion disrupts normal conduction with increased arrythmoge-nicity, which can be further aggravated by transplanting stemcells that do not differentiate into mature myocytes or intomyocytes that do not fully integrate with surviving myocar-dium.111 In most preclinical and clinical studies, stem cellshave been injected either intracoronary or intramyocardially.4

The expectation of these studies is that stem cells willdifferentiate into cardiomyocytes and would then align them-selves with surviving myocardium, with full electromechan-ical integration. As our knowledge on elongation and align-ment processes during development increases, new strategiesfor driving this integration may exploit biomaterials.111 Acrucial part of successful tissue engineering approaches isgraft/host integration of transplanted constructs. These con-structs not only need to be connected electrically and me-chanically to surviving myocardium but also need a connec-tion to the vasculature. Therefore, proangiogenic scaffoldsare promising materials for tissue engineering approaches.43

Development of tissue engineering strategies that promotealignment of newly formed myocytes with existing myo-cytes112 could decrease the arrythmogenicity of stem cell

transplantation and at the same time increase mechanicalcoupling.111 As described, nanotopography of biomaterialscan influence cell morphology and electromechanical inte-gration.97 Another strategy to increase adhesion is incorpora-tion of the common integrin adhesion peptide sequence RGD.For instance, RGD-labeled PEG and poly-lactic acid copoly-mers increase endothelial cell spreading in the polymer.113

Modification of polyether urethane (PEU) surfaces with RGDmoieties increased endothelial cell adhesion and viabilitywhen compared with PEU.114 However, RGD sequences arenot cell-specific and can enhance fibroblast adhesion aswell.10 Careful spacing and orientation of RGD ligands arealso important for control of cell spreading and proliferation;it has been shown that variations in nanoscale organization ofRGD in alginate gel altered the adhesion of preosteoblasts.10

Recently, a new concept of interaction between adultcardiomyocytes and progenitor cells has been proposed,mircrine signaling.115 Mircrine signaling entails diffusion ofmiRNA molecules through gap junctions between adjacentcells.115 The first described example is diffusion of miR-499from adult cardiomyocytes, which have high expressionlevels of miR-499, to neighboring cells.115 Theoretically,mircrine signaling could lead to coordination of gene expres-sion patterns between neighboring cells and a better electro-mechanical integration.

Cell Sheets and Engineered CardiacTissue Constructs

An alternative approach to injectable biomaterials is theepicardial implantation of in vitro engineered cardiac tissueconstructs.15 Patches are constructed in vitro by combining anartificial extracellular matrix with myocytes derived fromstem cells. A major advantage of this approach is that cellsare evenly distributed throughout the matrix and differentia-tion can occur in a controlled in vitro environment.15 Majordisadvantages are the need for surgical implantation and theneed for connection to the host vascular system for survival ofthe graft. Tissue-engineered constructs have been made withvarious biomaterials, including matrigel116 and collagen.51,116

We refer readers interested in this exciting research area tomore detailed reviews.13–15

Similarly to tissue-engineered patches, cells sheets of oneor more layers of in vitro-grown cells can be implantedepicardially after MI.117 The major difference between bothapproaches is that cell sheets have an extracellular matrixproduced by the cells themselves. Cell sheets of MSCsimprove cardiac function after MI118 by reversing cardiacwall thinning and improve animal survival after MI. In thesetransplanted MSCs, cell growth was observed, as was differ-entiation of a number of cells into cardiomyocytes and bloodvessels. The approach of cell sheet transplantation works onlyfor cells, like MSCs, that produce a strong collagenousmatrix. More immature stem cells with higher differentiationpotential do not produce enough extracellular matrix materialfor successful epicardial implantation.

Future DirectionsBiomaterials can enhance stem function by many differentmechanisms. In the near future, our knowledge of biomaterial




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engineering will increase in parallel with our knowledge ofnaturally occurring extracellular matrix and of stem cellbehavior. There is little question that molecularly designedbiomaterials can control many of the factors that guidedifferentiation and function of progenitor cells. The challengefor the cardiovascular field is to define robust and reproduc-ible progenitor biology so that the full power of modernbiomaterials can be exploited for therapeutic delivery.

Injectable hydrogels could be designed to control both thebiophysical and biochemical microenvironments of trans-planted cells. Furthermore, injectable hydrogels might lead toa more rapid translation into clinical cardiology. New tech-nologies have to be developed that allow for local drugdelivery together with transplanted stem cells and that allowfor tighter control of the microenvironment of transplantedcells. Delivery of multiple growth and differentiation factorstogether with different cell types in a spatially and temporallycontrolled manner will be needed for successful cardiacregeneration.

Sources of FundingSupported by grants from the National Institutes of Health(HL103582 and HL092930).

DisclosuresVincent F.M. Segers is a founder of and consultant for Provasculon.Richard T. Lee is a founder of and consultant for Provasculon.

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Vincent F.M. Segers and Richard T. LeeBiomaterials to Enhance Stem Cell Function in the Heart

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