Patching the Heart: Cardiac Repair from Within and Outside

Abstract

Transplantation of engineered tissue patches containing either progenitor cells or cardiomyocytes for cardiac repair is emerging as an exciting treatment option for patients with postinfarction left ventricular (LV) remodeling. The beneficial effects may evolve directly from remuscularization or indirectly through paracrine mechanisms that mobilize and/or activate endogenous progenitor cells to promote neovascularization and remuscularization, inhibit apoptosis, and attenuate LV dilatation and disease progression. Despite encouraging results, further improvements are necessary to enhance current tissue-engineering concepts and techniques and to achieve clinical impact. Herein, we review several strategies for cardiac remuscularization and paracrine support that can induce cardiac repair and attenuate LV dysfunction from both within and outside the myocardium.

INTRODUCTION

Heart failure often develops as a consequence of the limited endogenous capacity for cardiac regeneration after acute myocardial infarction (MI). ¹ Cardiac progenitor cells (CPCs) that have been transplanted into hearts with MI can repair the damaged myocardium to some degree, despite their low engraftment rates and limited evidence of transdifferentiation into myocardial cells. ^2-14 However, as progenitor- and stem cell-based tissue engineering technologies continue to evolve, it now appears possible to deposit and retain large numbers of therapeutic cells in close proximity to the region of myocardial tissue damage, and even to replace scar tissue, thus preventing scar bulging and LV dilation, reducing wall stress at the border zone (BZ) of the infarct, improving myocardial bioenergetics, and, finally, remuscularizing the heart. ^15-18

Tissue-engineered cardiomyoplasty, using engineered myocardium or cell sheets, was first introduced at the beginning of the millennium^19-25. Recently, the paracrine activity of the engineered tissue has been enhanced by using fibrin patches as the cell carrier. ^{16-18, 26} Importantly, evidence of myocardial salvage has been obtained with both the direct-remuscularization and paracrine approaches in rodent and porcine allograft models. ^{17, 23, 25} Paracrine factors led to significant increases in BZ vascular density and in the regeneration of myocytes from endogenous CPCs. Although these reports suggest that tissue-engineered patches could have therapeutic potential for the repair of myocardial injury, the overall engraftment rate and biophysical integration of grafts needs further optimization.

Engineering cardiac patches for heart repair is a challenging biotechnological objective. This review summarizes the progress in cardiac patch engineering (Table 1) and critically analyzes the problems and considerations that have to be incorporated into the design of an optimal engineered cardiac patch for heart-failure therapy. Despite the relatively straightforward biological rationale, the mechanisms responsible for the observed therapeutic effects are, in most cases, incompletely understood. Here, we review the available data on tissue-engineered heart repair and highlight evidence of the specific mechanisms of action associated with tissue-engineered patches.

Table 1.

Summary of Studies

		Cell Lines and Other Components	Reference	Summary/Observations
Natural Scaffolds	Collagen	Neonatal rat heart cells	Souren27,28	Floating collagen layers were used as a matrix for CM seeding and studies of myocyte contractile behavior
			Zimmermann29	First introduction of EHTs from neonatal rat CMs in collagen/Matrigel matrix mixtures and demonstration of the importance of mechanical loading
			Zimmermann30	3D EHTs show morphological, ultra-structural, and function properties of native myocardium and show distinct responses to growth factor stimulation
			Naito31	The non-myocytes and in particular fibroblasts are important for optimal EHT function and capillarization; Matrigel can be replaced by insulin and T3; fusion of individual EHTs can be employed to generate large tissue patches
			Tiburcy32	Immature CMs terminally differentiate and reach a postnatal degree of mature in EHTs
			Zimmermann20	EHTs are quickly capillarized and innervated after implantation
			Zimmermann23	Multi-loop EHTs can be used to enhance contractile performance of infarcted rat hearts
		Fetal rat heart cells	Li21	Cardiomyocytes can be seeded on gelatin sponge (Gelfoam); implanted grafts remain viable
		Chick embryo heart cells	Eschenhagen 37	First introduction of cardiomyocyte populated matrices (CMPMs) from embryonic chick cardiomyocytes in collagen gels
			Fink38	Stretching of EHTs induces hypertrophy and functional improvement
		Mouse embryonic stem cells	Guo39	Use of mouse embryonic stem cells as cardiomyocyte source for EHT construction demonstrated
		Mouse parthenogenetic stem cells	Didié40	Use of mouse parthenogenetic stem cells (PSCs) as cardiomyocyte source for engineered heart muscle (EHM) construction demonstrated; proof-of-concept for the utility of PSC-EHM in heart repair
		Human embryonic and induced pluripotent stem cells	Tulloch41	Human EHTs generated and cardiomyocyte proliferation observed
			Soong42	Human EHTs generated under serum-reduced conditions
			Streckfuss-Bömeke43	Different iPSC sources for cardiomyocyte derivation and EHT construction
		Rat MSCs	Maureira44	Contractility↑, perfusion↑, infarct size↓
	Fibrin	Neonatal rat vascular SMCs	Ross45	ECM gene expression correlates with tissue growth and development in fibrin gel
			Weinbaum46	Evaluates a method for monitoring collagen transcription in EHTs
		Neonatal rat cardiac cells	Black47	Cell alignment improves twitch force
		Human fibroblasts or rat SMCs	Long48	Elastogenesis can be achieved in tissue engineering
			Grassl49	Fibrin stimulates collagen production by SMCs
		miPSC-CMs	Liau50	Cardiac tissue derived from pluripotent stem cells has advanced structure and function
		Porcine MSCs	Liu26	Neovascularization↑, wall thickening fraction↑
		SDF-1α	Zhang51	Contractility↑, c-Kit+ cell homing↑
		HGF	Zhang52	Contractility↑, neovascularization↑, LV dilation↓, scar size↓
		hESC-ECs and hESC-SMCs	Xiong17	Contractility↑, neovascularization↑, bioenergetics↑, c-Kit+ cell homing↑, hypertrophy↓, apoptosis↓
		hiPSC-EC and hiPSC-SMC	Xiong18	Contractility↑. neovascularization↑, ATP turnover↑, c-Kit+ cell homing↑, hypertrophy↓, apoptosis↓
	Fibrin and matrigel	Neonatal rat cardiac cells	Hansen53	Introduces a simple system for constructing and evaluating EHTs
	Alginate	Neonatal rat cardiac cells	Dar54	Evaluates methods for optimizing cell seeding and distribution in 3D scaffolds
			Dvir55	Evaluation of a novel perfusion bioreactor that provides a homogenous milieu for tissue regeneration
		Fibroblasts	Shapiro56	Alginate may provide an excellent support for cell transplantation
		Fetal rat cardiac cells	Leor22	Contractility↑, angiogenesis↑, LV dilation↓
		Neonatal rat cardiac cells and gold nanowires	Dvir57	Gold nanowires promote conductivity
	Alginate and omentum	Neonatal rat cardiac cells	Dvir58	Contractility↑, angiogenesis↑, scar thickness↑, LV dilation↓
	Injectable alginate	Calcium-crosslinked alginate	Landa59	Contractility↑, LV dilation↓
		Peptide modified (without cells)	Yu60	Angiogenesis↑, LV function↑
		IGF-1 and HGF	Ruvinov61	Angiogenesis↑, infarct↓
	Gelatin gelfoam	Fetal rat cardiac cells	Li21	Cardiac graft survived and formed junctions with host heart cells
	Silk protein fibroin*	Neonatal rat cardiac cells	Patra62	Silk fibroin is suitable for cardiac-patch engineering
Synthetic Scaffolds	PGA	Neonatal rat cardiac cells	Carrier63	PGA introduced as synthetic material for myocardial tissue engineering
			Bursac64	Use of PGA seeded with cardiomyocytes as a model for cardiac electrophysiology
	Polyglycerol sebacate	Neonatal rat cardiac cells	Radisic65	Mathematical modeling of oxygen consumption
			Engelmayr66	Polyglycerol sebacate forms an accordion-like honeycomb scaffold that promotes heart-cell alignment and mechanical properties
	Injectable polyglycerol sebacate	Rabbit CMs	Ravichandran67	Evaluates a minimally invasive technique that uses injectable polyglycerol sebacate fibers for scaffold creation
	Poly ε-caprolactone	Mouse bone marrow cells	Fukuhara68	Angiogenesis↑, LV function↑
Decellularized hearts	PCLA	Rat SMCs	Matsubayashi69	Contractility↑, wall thickness↑, LV dilation↓
	Decellularized rat heart	Neonatal rat cardiac cells	Ott70	Evaluates the use of a decellularized matrix for engineering a bioartificial heart
	Decellularized heart	Human MPCs	Godier-Furnemont71	Contractility↑, LV dilation↓
	Decellularized porcine bladder	hESC-ECs, HUVECs, mouse CMs	Turner72	Evaluates the use of a decellularized matrix for delivering ESC-derived cardiac cells
Cell Sheets		Neonatal rat ventricular CMs	Haraguchi73	Electrical coupling of CM sheets occurs rapidly
			Sekiya74	Bioengineered cardiac cell sheets have intrinsic angiogenic potential
			Hata75	Evaluates a 3D cardiac patch made of cell sheets and decellularised matrix
			Shimizu19	Spontaneous, macroscopic beating is observed in subcutaneously implanted sheets
			Shimizu76	Subcutaneously implanted sheets survive for at least one year
		Neonatal rat CMs and rat ECs	Sekine77	Contractility↑, angiogenesis↑, fibrosis↓
		Rat adipose-derived MSCs	Miyahara24	Contractility↑, scar size↓, LV dilation, mortality↓
		Monkey stromal cells and monkey SSEA-1 and CPCs	Bel78	Angiogenesis↑, cell engraftment rate↑
		Bovine aortic endothelial cells	Kushida79	Evaluates a temperature-responsive surface for culturing cell sheets
		mESC-derived cardiac cells	Masumoto80	Contractility↑, wall thickness↑, angiogenesis↑, fibrosis↓
		hESC-CMs	Stevens81	Evaluates a novel scaffold-free human myocardial patch made of hESC-CMs
		hESC-CMs and ECs	Stevens82	Cell engraftment↑
		hiPSC-CMs	Matsuura83	hiPSC-CMs can be used to create thick cardiac tissues
			Lee84	Evaluates a multiparametric imaging system that simultaneously measures action potentials and intracellular calcium-wave dynamics of cardiac cell sheets
			Kawamura25	Contractility↑, angiogenesis↑, LV dilation↓

3D: three-dimensional; CM: cardiomyocyte; CPC: cardiac progenitor cell; EC: endothelial cell; ECM: extracellular matrix; EHT: engineered heart tissue; hESC: human embryonic stem cell; HGF: hepatocyte growth factor; hiPSC: human induced-pluripotent stem cell; HUVEC: human umbilical-vein endothelial cell; IGF-1: insulin-like growth factor 1; LV: left ventricuar; miPSC: murine induced-pluripotent stem cell; MPC: mesenchymal progenitor cell; MSC: mesenchymal stem cell; PCLA: ε-caprolactone-co-L-lactide reinforced with knitted poly-L-lactide; PDGF-BB: platelet-derived growth factor BB; SDF-1: stromal cell-derived factor 1; shRNA: small-hairpin RNA; SkM: skeletal myoblast; SMC: smooth muscle cell; SSEA-1: stage-specific embryonic antigen 1; VEGF: vascular endothelial growth factor.

PATCHING THE HEART WITH TISSUE-ENGINEERED MYOCARDIUM

Generating functional bioengineered human cardiac tissue for the treatment of end-stage heart disease has been a tremendous challenge for researchers and physicians alike. Tissue engineering was first proposed as a means to rebuild organs for therapeutic applications in the early 1990s⁸⁵ and later extended to the cardiac field.^{20-22, 86} Generally, bioengineered cardiac tissue constructs can be divided into two categories: scaffold-based and scaffold-free. A number of three-dimensional (3D) scaffolds have been fabricated from natural biological materials, such as collagen, fibrin, and alginate, from naturally occurring extacellular matrix mixtures (Matrigel™) or decellularized heart matrix, and from synthetic polymers.

SCAFFOLD-BASED APPROACHES

Collagen

Collagen is the most prevalent extracellular component of the myocardium and has been used as a matrix for studying myocardial electrophysiology and contraction since the early 1990s^{27, 28}. It is self-polymerizing at 37°C and physiological pH, can be molded into a variety of shapes, and when seeded with heart cells, the resulting matrix possesses many of the physiological characteristics of cardiac tissue^{29, 30, 32, 37, 41, 87}. A key property of collagen hydrogel-based engineered heart muscle (EHM) is its contractile performance, which reproduces many, but not all, aspects of contractile performance in native myocardium^{29, 30, 37}. The first EHM produced from mammalian (in this case, neonatal rat heart) cells was generated with a combination of collagen and Matrigel™ ²⁹. Mechanical loading was subsequently identified as an important factor for tissue maturation^{29, 38}, and direct comparisons with the development of native heart tissue identified organotypic maturation in collagen-based EHM that possessed the capacity to support hypertrophic growth³². Implantation of collagen-based EHM promoted further maturation, which was paralleled by vascularization and innervation^{20, 23}.

One week after transplantation onto uninjured rat hearts, collagen scaffolds containing cardiomyocytes that had been generated through the controlled differentiation of human embryonic or induced-pluripotent stem cells (hESC-CMs or hiPSC-CMs, respectively), endothelial cells (ECs), and stromal cells contained vascular structures derived from the transplanted human cells and were perfused by the host animal’s coronary circulation.⁴¹ In a rat MI model, transplantation of a collagen scaffold containing mesenchymal stem cells (MSCs) was associated with improvements in infarct size, ventricular wall thickness, angiogenesis, perfusion, and contractile function, and with increases in the growth of myofibroblast-like tissue⁴⁴. Collagen scaffolds have also been used to deliver vascular endothelial growth factor (VEGF) both directly, by modifying the protein to include a collagen-binding domain⁸⁸, or indirectly, by seeding the scaffold with cells that have been genetically engineered to express VEGF⁸⁹, thereby extending the duration of protein delivery and promoting vascular growth in infarcted myocardial tissue⁸⁸.

The fusion of individual EHM rings into multi-loop constructs enables the creation of engineered tissues with, in principle, unlimited dimensionality (Figure 1)³¹. Implantation of a multi-loop patch two weeks after MI in rats led to sustained therapeutic benefits with enhanced regional contractility 4 weeks after engraftment²³. Furthermore, an alternative pattern of EHM ring assembly can be used to generate cardiac-tissue pouches that provide restraint and contractile support to failing hearts (Figure 1)³³, and in-vitro studies demonstrate that EHM rings can serve as a test bed for receptor pharmacology ²⁹, growth factor signaling ^{30, 34}, and target validation through the use of adenoviral transduction ³⁵ and hypertrophic signaling ^{32, 90}. hESC- and hiPSC-CMs can also be used to generate collagen-based EHM^{41, 87, 91}, and parthenogenetic stem cells have recently been introduced as another source of stem cells for cardiomyocytes and myocardial tissue engineering ⁴⁰. Further advances in collagen-based myocardial tissue will likely involve biomimetic culture platforms that incorporate electrical³⁶ and mechanical stimulation²⁹.

Figure 1.

Applications of engineered heart muscle (EHM). Schematic presentation of (A) an EHM patch and (B) an EHM pouch (red). (C) Illustration of a multi-loop EHM with five loops fused together to form an asterisk-shaped stack with a solid center surrounded by 10 loops that are used for surgical fixation of the EHM graft. (D) The patch was fixed over the site of infarction in rats with six single-knot sutures. (E) The dimensions of an EHM pouch and an explanted rat heart are shown. (F) An EHM pouch was implanted over a healthy rat heart to simulate its use as a biological ventricular assist device. Panels C and D are modified from Zimmermann et al. Nat Med. 2006; 12(4): 452-8²³; panels E and F are modified from Yildirim et al. Circulation 2007;116(11 Suppl): I16-23³³.

Fibrin

Fibrinogen is cleaved and cross-linked by exposure to thrombin and clotting factor XIII to form an insoluble fibrin mesh that captures blood cells to form a blood clot. Fibrin meshes can also be used to capture any target cell for tissue engineering. In vivo, fibrin attracts leukocytes and, in particular, macrophages, and is slowly lysed by endogenous proteases. Anisotropic alignment of rat neonatal cardiac cells in fibrin scaffolds was associated with increases in twitch force, despite declines in total collagen and protein content⁴⁷. Fibrinogen can also be conjugated with polyethylene glycol (PEG) to generate scaffolds that covalently bind growth factors and other proteins^{51, 52}, and drug screening platforms have recently been established with fibrin-based engineered heart tissue^{53, 92}. In a mouse MI model, treatment with a PEGylated fibrin patch containing stromal cell-derived factor-1 (SDF-1) was associated with increases in CPC recruitment and improvements in infarct size and cardiac function; the patch was placed over the site of infarction, where it was associated with increases in SDF-1 levels for up to 28 days⁵¹.

Fibrin patches with defined biophysical properties can be created by mixing solutions of fibrinogen and thrombin, and because the mixture typically solidifies in less than one minute, the patch can be created in situ by injecting the two solutions into a mold placed over the infarct site. This method has been used to deliver autologous MSCs to swine²⁶, and hESC-derived vascular cells (hESC-VCs) (i.e., smooth-muscle cells [SMCs] and ECs) to both swine and mice^{17, 18, 24}, after experimentally induced MI. The transplanted MSCs differentiated into myocyte-like cells⁸ and were associated with greater thickening of the infarcted wall during contraction, while the hESC-VC treatments were associated with improvements in myocardial function, perfusion, and energy metabolism, and with declines in left-ventricular wall stress and remodeling.⁹³ Furthermore, the proportion of cells retained in the ischemic region was significantly higher when the cells were administered via a patch than when injected directly into the myocardium.⁹³ Thus, a fibrin patch can be used as a unique platform for cell delivery.

Other 3D Biological Scaffolds

Other 3D biomaterials that have been used to engineer myocardial tissue equivalents include alginates^{54, 56} and silks⁶². However, preformed matrices do not appear to be ideally suited for the assembly of cardiomyocytes into a functional 3D cardiac syncytium of contracting cells because the rigid structures/pores of the matrix isolate the cardiomyocytes from each other, thereby preventing direct intracellular contact. This caveat also applies to preformed collagen/gelatin sponges; however, gold nanowires have been used to improve the synchronous excitation of engineered cardiac tissue by bridging the electrically resistant pore walls of alginate.⁵⁷ In addition, it seems essential that the exogenous matrix be rapidly replaced to prevent interference with tissue formation. Vascularization of the matrix occurs quickly in conjunction with the immune response to the presence of foreign materials.

Decellularized Matrix

Recellularization of a previously decellularized rat heart was successfully attempted in 2008, which suggests that whole organs can be reengineered by using a natural ECM blueprint⁷⁰. Natural scaffolds made from decellularized porcine small-intestinal submucosa (SIS)⁷⁵ and bladders⁷² and from human myocardial tissues⁷¹ have been used to generate 3D cardiac patches. For example, decellularized SIS has been layered with multiple sheets of neonatal rat cardiomyocytes to produce patches of up to 800-μm thickness, resulting in cardiac muscle constructs that contracted synchronously for up to 10 days⁷⁵. Cell sheets broke apart when applied to decellularized bladder matrix, presumably because of the roughness of the matrix surface, but remained intact and proliferative when the voids of the matrix were filled with hyaluronan hydrogel; the hydrogel also enabled a greater number of cells to be seeded. Hydrogels made of fibrin and containing suspended human mesenchymal progenitor cells have been applied to decellularized sheets of human myocardium⁷¹ to create cardiac patches that were subsequently evaluated in a nude-rat model of MI. The patches dramatically increased both the recruitment of mesenchymal progenitor cells and vascular growth in the infarcted region, and measurements of contractility and left ventricular (LV) dimensions were similar to those obtained in uninjured hearts; integration of the patch with the myocardium of the recipient heart was not examined.

Synthetic Polymers

Polygycolic acid (PGA)⁶⁸, poly ε-caprolactone-co-l-lactide (PCLA)⁶⁹, poly-glycerolsebacate (PGS)⁶⁶, and polydimethylsiloxane (PDMS)⁹⁴ have been extensively used as tissue engineering substrates. These materials typically must be coated with extracellular matrix proteins to enable cardiomyocyte attachment and have many of the same limitations associated with biological matrices (as described above) for heart-muscle engineering on a macroscopic scale. However, they may be well suited for depositing cells with paracrine activity onto the heart and for providing structural support to prevent further ventricular dilatation.

Injectable Systems

Injectable scaffolds have been used to prevent LV wall thinning and bulging of the ischemic myocardium, to inhibit LV remodeling⁵⁹ and to promote angiogenesis by recruiting endogenous CPCs^{60, 61}. The injection of a cell-free, in situ–forming, alginate hydrogel into recent (7 days) and old infarcts (60 days) provided a temporary scaffold that attenuated adverse cardiac remodeling and improved contractility, and the benefits were comparable to those obtained with the intramyocardial injection of neonatal cardiomyocytes⁵⁹. Angiogenesis can be enhanced by integrating RGD motifs into an alginate hydrogel⁶⁰, and similar results were obtained with alginate scaffolds that had been loaded with insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF)⁶¹. In a rat model of acute MI, intramyocardial injections of the affinity-bound IGF-1/HGF alginate biomaterial increased angiogenesis and mature blood vessel formation at the infarct, preserved scar thickness, attenuated infarct expansion, and reduced scar fibrosis after 4 weeks. In an alternative approach, cardiomyocyte retention was improved when the cells were co-injected with nanofibers made of poly-glycerolsebacate (PGS)⁶⁷.

SCAFFOLD-FREE APPROACHES

The development of scaffold-free cell sheets first became practical after the invention of a temperature-responsive polymer substrate, poly-(N-isopropylacrylamide) (PIPAAm)⁹⁵. Cardiomyocyte cell sheets are grown on the polymer, released by a drop in temperature, and then stacked to yield multilayered tissues^{19, 79, 96}. Electrical connections between cardiomyocyte sheets form quickly, probably because, in contrast to enzymatic sheet release, the temperature responsive plates leave all surface protein structures intact, thus facilitating gap junction formation⁷³. Capillary growth can be induced by creating stacks composed of both cardiomyocyte and ECs^77,74, and engrafted cell sheets survived and remained contractile for up to 1 year after implantation⁷⁶. Other scaffold-free tissue engineering approaches include the construction of cardiac microtissues by using a hanging drop method or spontaneous aggregation.^{81, 97} Both cell-sheet and cell-aggregation technologies have been successfully used with cardiomyocytes derived from pluripotent stem cells^{25, 80, 81, 83, 84}, and recently, composite cell sheets consisting of monkey adipose-derived stromal cells and monkey ESC-derived SSEA-1⁺ cells were tested in vivo with encouraging results⁷⁸. Thus, cell sheet-based cardiac tissue engineering may prove to be a useful strategy for cardiovascular tissue repair that suppresses LV dilatation, increases wall thickness, and improves LV chamber function.

MYOCYTE TURNOVER

The concept of cardiomyocyte turnover in the adult heart is relatively new, and whether the limited regenerative capacity of the adult heart is mediated by the proliferation of pre-existing cardiomyocytes or through the activity and derivatives of endogenous CPCs remains a matter of intense debate. ^98-108 Several markers can be used to identify endogenous CPCs with cardiogenic potential, including c-Kit^{2, 3}, Sca-1^{14, 109}, Abcg2¹¹⁰ and Isl1 ^{111, 112}. Once the types of CPCs are fully characterized, and their pathways of activation are deciphered, biomaterial patches can be designed to promote CPC activation and cardiomyocyte turnover, and then placed over the site of myocardial injury, leading to the more comprehensive activation of mechanisms for myocyte regeneration both within and outside the myocardium.

PATCH-ASSOCIATED PARACRINE SUPPORT in MYOCARDIAL PROTECTION and the ACTIVATION OF ENDOGENOUS REPAIR MECHANISMS

Cell Patch- and Paracrine Support-induced Myocardial Repair from Endogenous Cardiac Progenitor Cells

Although CPCs that have been transplanted into hearts after MI can repair the damaged myocardium to some degree, this activity is limited because the engraftment rate is low and trans-differentiation of the engrafted cells occurs even less frequently.^4-10 Thus, myocyte regeneration from the transplanted cells has been suboptimal^11-14, and additional benefits might be obtained by applying a fabricated cell patch over the periscar BZ to mobilize endogenous repair mechanisms, stabilize the chronically evolving infarct scar, reduce BZ wall stress, and improve myocardial bioenergetics. The Zhang group used a fibrin patch to enhance delivery of hESC-VCs for myocardial repair. ¹⁷ The hESC-VCs that were loaded into the patch released a variety of cytokines that promote angiogenesis and survival, and reduce apoptosis. ^{17, 93} Both in vitro and in vivo experiments demonstrated that the hESC-VCs effectively inhibited myocyte apoptosis, and the patch-enhanced delivery of hESC-VCs alleviated abnormalities in BZ myocardial perfusion, contractile dysfunction, and LV wall stress. These results were also accompanied by the pronounced recruitment of endogenous c-kit⁺ cells to the injury site, and were directly associated with a remarkable improvement in myocardial energetics, as measured by a novel in vivo ³¹P magnetic resonance spectroscopy method. Similar findings were obtained with fibrin patches containing VCs derived from human induced pluripotent stem cells (hiPSCs) ¹⁸. Four weeks after MI and treatment, LV structural and functional abnormalities were less severe in hearts that received the patch-enhanced celltherapy, and these improvements were accompanied by significant reductions in infarct size¹⁸. hiPSC-VC transplantation also mobilized endogenous progenitor cells into the BZ, attenuated regional wall stress, stimulated neovascularization, and improved BZ perfusion, which led to marked increases in BZ contractile function and ATP turnover rate¹⁸.

Cytokine-associated Myocardial Protection and Increased Density of Myocardial Resistant Vessels

It is a rather consistent finding in the literature that,1) only a very small percentage of transplanted cells show long term engraftment in the recipient myocardium^{8, 113, 114}, and 2) that an even smaller fraction of the engrafted CPCs trans-differentiate into cardiomyocytes or VCs^{8, 113, 114}. These findings have led to the belief that early paracrine interactions between the transplanted cells and native cardiomyocytes, VCs, and/or (possibly) cardiac and vascular progenitor cells provide much of the benefit associated with cell transplantation^{115, 116}. Indeed, many reports indicate that cell transplantation performed shortly after an ischemia-reperfusion (I-R) event is associated with early declines in apoptosis of the injured cardiomyocytes. This sparing of native cardiomyocytes that would otherwise have died after the initial I-R insult likely reduced the size of the infarct and led to the subsequent declines in LV remodeling and dysfunction that were observed in cell-treated animals^{8, 10}. Furthermore, evidence reported by Yamashita’s group indicates that cardiomyocytes may be the main source of VEGF in cardiac-tissue sheets⁸⁰; they found that the omission of cardiomyocytes from transplanted sheets led to the disappearance of neovascularization and functional improvement, suggesting that the beneficial effects of the sheet were induced by cardiomyocyte-secreted cytokines.

Although these findings support the view that paracrine effects initiated by the engrafted cells contribute to declines in infarct size by decreasing apoptosis when the therapy closely follows the ischemic event, cardiomyocyte apoptosis may also be reduced at later time points, and if so, this decline may result from the somewhat delayed increase in BZ capillary density after cell-patch transplantation^{8, 10, 117}. The patch-enhanced delivery of hESC-VCs and hiPSC-VCs was accompanied by significant improvements in myocardial perfusion in both the infarct and the BZ as well as a significant increase in the number of resistant vessels in the myocardium, which suggests that vessels generated by the transplanted, stem-cell derived VCs were functional^{17, 18}. Furthermore, these vessels are the smallest muscular arterioles (50 ~150 μm) that regulate myocardial perfusion, and each arteriole supports capillaries where the exchange of oxygen and carbon dioxide occurs. Thus, since it has been previously reported that LV hypertrophy and failure is associated with subendocardial ischemia during elevated cardiac work states^{118, 119}, this increase in resistant-vessel density could provide the structural basis for the increases in myocardial blood flow, declines in apoptosis, and improvements in LV contractile performance that have been observed in preclinical and clinical studies^{17, 120-122}.

CONCLUSION and FUTURE WORKS

Acute myocardial damage is exacerbated by chronic myocardial overload and LV dilatation, which often leads to heart failure. Consequently, the goal for future regenerative/reparative cardiac therapies includes at least two components: 1) minimizing oxidative myocardial damage and remodeling secondary to LV chamber dilatation, and 2) rebuilding the muscle of infarcted and chronically failing hearts to improve contractile performance. Although the paracrine mechanisms induced by cytokine administration may be well suited for delaying and preventing pathological remodeling and disease progression, the repair of significant ventricular scar bulging will likely require the generation of new cardiac muscle. Thus, strategies that combine the administration of paracrine factors with the creation and integration of engineered cardiac tissue are particularly attractive for the treatment of LV dilatation in hearts with postinfarction LV remodeling.

The beneficial effects of engineered cardiac tissue patches have been clearly demonstrated in animal models, and the clinical feasibility of this approach is supported by the successful transplantation of collagen sponges that lack cardiomyocytes^{123, 124} and of cell sheets that contain skeletal myoblasts¹²⁵. Nevertheless, cardiac patches have yet to be extensively investigated in clinical studies, primarily because suitable sources for human cardiomyocytes are lacking. The availability of human stem cells, including hiPSCs, and highly efficient protocols for directing differentiation^{15, 126, 127} could alleviate this scarcity and may enable patients to be treated with patches engineered from their own cells,^{15, 127} thereby minimizing the potential complications that could evolve from the induction of the immune and inflammatory responses. However, the time required to generate hiPSC-CMs precludes their use in patients who need prompt treatment; thus, many patients who are candidates for cardiac-patch therapy will benefit from the continued development of patches that contain allogenic cell lines.

Cardiac patch therapy can only be successful if the patch quickly becomes integrated and perfused by the recipient’s coronary circulation. Although current tissue engineering platforms are rapidly vascularized, and many interventions can successfully promote neovascularization in the myocardium over time, the vessels may not develop quickly enough to prevent a significant fraction of the cardiomyocytes in the patch from initiating necrotic processes and apoptosis, which can occur within 30 minutes of exposure to no flow ischemia. Furthermore, the engineered tissue is less mature than the recipient myocardium, so endogenous mechanisms for sensing and responding to hypoxic conditions within the patch may not be activated by the absence of immediate vascular support. Thus, the integrity and function of the transplanted patch may be improved by the addition of factors that stimulate vessel growth, impede apoptosis, and promote graft maturation.

Engineered cardiac-tissue patches possessing both contractile and paracrine activity have been successfully applied in acute and subacute models of myocardial injury, and patches designed to promote CPC activity and/or cardiomyocyte turnover could be placed over the injury site to stimulate cardiomyocyte regeneration both at the myocardial surface and within the tissue (Figure 2). This strategy can also be extended to include the creation of engineered cardiac-tissue pouches that provide paracrine and contractile support over the entire ventricular surface (Figure 1). However, the clinical acceptance of any patch-based therapy will require the development of a practical and minimally invasive delivery method; for example, an endoscope/catheter-based system could be used to access the pericardial sac through the abdomen and diaphragm, and then solutions could be injected through the catheter to form a hydrogel-based patch in situ. Collectively, these advancements could lead to the development of a new generation of patch-based cellular therapies that combine paracrine support and remuscularization to promote cardiac repair both from within and from outside the myocardial tissue.

Figure 2.

The benefits associated with our in vivo method for cardiac patch application. A circular 3D porous biodegradable cardiac patch (blue) is created over the infarcted region by mixing thrombin and fibrinogen solutions that contain different progenitor cell types, such as mesenchymal stem cells (MSCs), vascular cells (VCs) generated through the controlled differentiation of either human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), or both hiPSC-VCs and cardiomyocytes (hiPSC-CMs). The fibrinogen can be modified to bind peptides for different purposes, such as guiding differentiation or impeding apoptosis. The solution typically solidifies in less than one minute to form a 3D porous cardiac patch that provides structural support as well as a platform for the transplanted progenitor cells, and ultimately increases the cell engraftment rate. The transplanted cells release growth factors and other cytokines that reduce apoptosis, promote angiogenesis, and activate endogenous mechanisms for cardiomyocyte renewal, which leads to declines in infarct size and to improvements in myocardial perfusion, metabolism, and contractile function. Measurements of in vivo myocardial bioenergetics and the ATP turnover rate (via ³¹P magnetization saturation transfer) suggest that the patch also protects against adverse changes in cardiomyocyte energy metabolism, perhaps by reducing wall stress and bulging at the site of the infarction. Collectively, these benefits improve cardiac contractile function and impede the progression of LV dilatation. (Panels B and K courtesy of Xiong Q et al Circ Res. 2012 ;111(4):455-68; Panels F, G, I, J and L, courtesy of Xiong Q et al Circulation 2013; 127(9): 997-1008).

Non-standard Abbreviations and Acronyms

3D

three dimension

Abcg2

ATP-binding cassette sub-family G member 2

BLI

bioluminescent imaging

BrdU

bromodeoxyuridine

BZ

border zone

CHF

congestive heart failure

CPC

cardiac progenitor cell

c-Kit

stem cell growth factor receptor

EC

endothelial cell

ECM

extracellular matrix

EHM

engineered heart muscle

ESC

embryonic stem cell

GFP

green fluorescent protein

HEP

high energy phosphates

hESC

human embryonic stem cell

hESC-CM

human embryonic stem cells derived cardiomyocyte

hESC-VC

human embryonic stem cells derived vascular cell

HGF

hepatocyte growth factor

hiPSC-CM

human induced pluripotent stem cells derived cardiomyocyte

IGF

insulin-like growth factor 1

I/R

ischemia reperfusion

I-R

ischemia-reperfusion

IZ

infarct zone

Luc

firefly luciferase

LV

left ventricular

MI

myocardial infarction

MRI

magnetic resonance imaging

MRS

magnetic resonance spectroscopy

MSC

mesenchymal stem cell

PCL

poly ε-caprolactone

PCLA

poly ε-caprolactone-co-l-lactide

PDGF-BB

platelet derived growth factor-BB

PDMS

polydimethylsiloxane

PEG

polyethylene glycol

PGA

polygycolic acid

PGF

poly(glycerol sebacate)

PGS

poly-glycerol-sebacate

PIPAAM

poly-(N-isopropylacrylamide)

RGD

arginine-glycine-asparagine

RZ

remote zone

Sca-1

stem cell antigen-1

SDF-1

stromal cell-derived factor-1

SIS

small-intestinal submucosa

SkM

skeletal myoblasts

SMC

smooth muscle cell

SSEA-1

stage-specific embryonic antigen-1

VC

vascular cell

VEGF

vascular endothelial growth factor