Stem Cell-Based Cardiac Tissue Engineering
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- Nunes, S.S., Song, H., Chiang, C.K. et al. J. of Cardiovasc. Trans. Res. (2011) 4: 592. doi:10.1007/s12265-011-9307-x
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Cardiovascular diseases are the leading cause of death worldwide, and cell-based therapies represent a potential cure for patients with cardiac diseases such as myocardial infarction, heart failure, and congenital heart diseases. Towards this goal, cardiac tissue engineering is now being investigated as an approach to support cell-based therapies and enhance their efficacy. This review focuses on the latest research in cardiac tissue engineering based on the use of embryonic, induced pluripotent, or adult stem cells. We describe different strategies such as direct injection of cells and/or biomaterials as well as direct replacement therapies with tissue mimics. In this regard, the latest research has shown promising results demonstrating the improvement of cardiac function with different strategies. It is clear from recent studies that the most important consideration to be addressed by new therapeutic strategies is long-term functional improvement. For this goal to be realized, novel and efficient methods of cell delivery are required that enable high cell retention, followed by electrical integration and mechanical coupling of the injected cells or the engineered tissue to the host myocardium.
KeywordsCardiac tissue engineeringStem cellsCell therapyRegenerative medicineInduced pluripotent stem cellsEmbryonic stem cells
Myocardial infarction (MI) occurs when one or more of the blood vessels supplying the heart are occluded. The blockage of coronary arteries leads to a sudden decrease in the supply of nutrients and oxygen to the portion of heart muscle supplied by the artery. When blood supply is not reestablished in time, irreversible cell death within the affected part of the heart muscle will occur. Such injuries to the myocardium result in the formation of scar tissue that does not have contractile, mechanical, or electrical properties of normal myocardium. By consequence, cardiac output is reduced, decreasing the ability of the heart to supply blood efficiently. The deterioration of heart function will lead to heart failure, and at the end-stages, the only treatment options are heart transplants and ventricular assist devices [1, 2]. Therefore, new therapies are required to prevent the progression of pathological remodeling and cell death, as well as to induce tissue recovery in the affected areas.
Regenerative medicine aims to achieve this goal through the restoration of tissue structure and organ function, thus delaying or preventing disease progression . In the case of therapies for heart disease, the desired outcome would be maintenance of normal ventricular function and anatomy , replacement of cardiomyocytes lost post-MI, prevention of left ventricular wall thinning, and improvement in overall cardiac output towards physiological levels (average of 5.6 L/min for human males and 4.9 L/min for human females) . Therefore, cell-based therapies represent a potential cure for patients with cardiac diseases such as MI, heart failure, and congenital heart diseases. Towards this goal, cardiac tissue engineering is now being investigated as an approach to support cell-based therapies and enhance their efficacy. In a classical approach, cardiac tissue engineering involves the integrated use of cells, biomaterials, and bioreactors with the purpose of generating a contractile myocardium .
Cardiac Tissue-Engineering Strategies
Direct Cell Injection Strategies
Since MI may result in loss of up to one billion cardiomyocytes in the infarct zone, the idea of regenerating myocardium by cell injection has emerged  and was tested in a number of notable in vivo studies over the past 20 years. Although direct cell injection into the heart does not result in a piece of beating cardiac tissue such as in conventional in vitro cardiac tissue engineering approaches, we classify it here as tissue engineering since it enables cardiac regeneration through application of cells and occasionally biomaterials. Initially, it was thought that injection of beating cells is required for restoration of the function. However, a large body of evidence suggests functional improvements even with the injection of non-contractile cells such as bone marrow cells or endothelial progenitor cells . Of note, while not the focus of this review, others have demonstrated efficacy of injecting biomaterials alone , such as decellularized matrices , in improving cardiac function.
In vivo cell injection studies in myocardial infarction model
SD rat (M)
Comp. LAD ligation
hESC (H9.2)-derived CM
1.5 × 106
71% TnI + (ICC)
4 and 8
FS ↑, LVDd ↓ (echo)
α-actinin + (IHC), no teratoma
Lewis rat (F)
Comp. LAD1 ligation
2 × 106
2 and 6
LVDA ↓ (echo)
Some TnI + (IHC)
Wall thickness ↑
C57BL/6 or AN mice (M)
Comp. LAD ligation
Fibroblast or iPS
0.2 × 106
EF↑ , LVDd ↓ (echo) wrt. fibroblast
α-Actinin + (IHC), no teratoma
Heart Size ↓
SVE129 mice (F)
Comp. LAD ligation
D3 mESC-derived Nkx2.5 + CPC
0.5–1 × 106
LVDd↓, EF ↑ (echo)
MHC + (IHC)
Infarct size ↓ (IHC)
Comp. LAD ligation
Tet Notch mESC- derived CM
0.5 × 106
2 and 8
EF↑ at 2 week (MRI)
TnT + (IHC)
Comp. LAD ligation
3 × 106
No improvement (MRI)
Comp. LAD ligation
Human fetal sca-1+ and -derived CM
0.5 × 106
70% TnI+ (ICC)
EF↑, EDV↓ (MRI)
5% Retention, 50% TnI + from both CP and CM (IHC)
Wall thickness ↑ (MRI)
Biomaterial with or without cells
SD rats (M)
Comp. LCx ligation
EF↑ (P-V catheter)
Infarct size ↓
Athymic SD rats (m)
Temp. LAD ligation
hESC (H7)-derived CM
1 × 107
PSC + GFR-matrigel
8, 16, and 32
Grafts were separated by scar tissue (IHC)
Temp. LAD ligation
hESC (H7)-derived CM
1 × 107
PSC + matrigel
LVEDD ↓, EF ↑(echo, MRI)
TnI + (IHC)
SD rats (F)
Comp. LAD ligation
1 × 107
ES/D D↓, EF↑ (echo)
Mixed TnT + and TnT-cells
Infarct size ↓
There were profound differences in the type of animal MI model and the cell injection time point, which rendered direct comparisons of the results difficult. Among the studies reviewed here, another significant difference was that all the studies with biomaterial injections (with or without cells) were performed many days after an MI using rat model, as opposed to those with cell injections, where most report injection of cells immediately post-MI using mouse model. This might be due to difficulties in producing large amounts of cells for injection; however, the difference in cell injection time point between these two animal models makes comparison of the outcome more difficult.
Some studies investigated functional integration between the injected cells and the host cardiomyocytes by immunostaining for cell junctional molecules such as connexin 43 or cadherin (Table 1). However, none of the studies showed any significant integration. The study conducted by Fernandes et al.  indicated that grafts were separated by scar tissue from the host myocardium, which may be a result of a natural process occurring with foreign cell injections. Current studies indicate that injecting biomaterials with or without cells has a distinct beneficial effect on decreasing infarction size and improving wall thickness. However, the mechanism responsible for functional improvements as a result of injection of biomaterials alone is yet to be elucidated. One possible explanation is that the injection of biomaterials results in mechanical stabilization of the ventricle irrespective of effects on cellular function or survival .
To facilitate further comparison between different studies, it appears necessary to standardize the experimental conditions and MI models. We believe that MI models where injections are performed at a later time point after the induction of MI are more clinically relevant since they likely reflect more closely the human MI scenario than models in which MI is induced and cells are injected right away. In addition, long-term studies maybe necessary to ensure that the functional improvement is not transient. Also, the generation of new in vitro systems which allows for the systematical testing of many important parameters such as functional integration between injected and host cells would be beneficial.
Direct Tissue Replacement Strategies
The fact that some improvement in function is observed regardless of the cell type injected and that cell retention is very small, indicates that improvement in cardiac function is likely due to the secretion of paracrine factors by the injected cells [25, 26] resulting in an increased preservation of affected myocardium in a transient manner as opposed to transdifferentiation and direct integration of contractile cells [26, 27]. In addition, the unlikely possibility that injected cells would, on their own, recapitulate the complex tissue organization present in cardiac muscle where cardiomyocytes, vascular, and stromal cells are positioned together in an intricate organization has increased the drive for new direct replacement tissue-engineering strategies. The objective of cardiac tissue engineering for direct replacement is to generate tissue constructs or mimics that can functionally replace damaged myocardium. This requires the use of biocompatible and/or biodegradable materials that serve as a scaffold for tissue mimics . In order to generate cardiac tissue mimics, a large number of cells are needed to ensure proper tissue function and integration when implanted in the affected areas.
In adults, the ability to repair damaged cardiac tissue is hindered by the reduced proliferative capacity of mature cardiomyocytes . For true myocardial regeneration, both the beating myocardium and the functional vasculature need to be regenerated in the infarct zone. Pluripotent stem cells, such as ESC and iPS cells, hold the potential to differentiate into all cell types in the body  and have the potential to be propagated in vitro for long periods of time. By consequence, these cells are a useful tool to generate large numbers of cells that hold the potential to not only exert functional benefits by secretion of paracrine factors but also to enhance cardiac function through differentiation and direct integration into cardiac tissue. However, safety issues involving differentiation into non-cardiac cell types or even formation of teratomas , hinder direct clinical translation at this time. Therefore, another possibly safer source of autologous cardiovascular cells is adult stem cell such as resident cardiac progenitors. However, such cells are usually present in very low numbers in adults.
Embryonic Stem Cells
Many different techniques have been used to assemble cardiac tissue mimics, from scaffold-free self assembled cells [31, 32] to the use of different biocompatible and/or biodegradable materials as scaffolds (for a review, see ). Grafting of mESCs cultured in polyglycolic-acid biodegradable scaffolds into infarcted mouse myocardium significantly improved animal survival, blood pressure, and ventricular function . Authors also report the presence of implanted cells in the infarcted area suggesting cell retention and possible myocardium repair . Other studies utilizing injection of undifferentiated ESC reported the formation of teratomas suggesting that this approach is not clinically relevant .
In order to generate biomechanical support and cell delivery to the heart, Chen et al.  generated hybrid cardiac patches made with poly(glycerol sebacate) and supplemented with hESC-derived cardiomyocytes. These patches sustained cell beating for long periods in culture and, when sutured over the left ventricle of normal rats, remained intact without deleterious effect on ventricular function, suggesting that these patches could function as support devices for cardiac repair. In addition, the delivery of stem cells in poly(glycerol sebacate) could potentially be more effective by utilizing an accordion-like honeycomb microstructure since Engelmayr et al. have demonstrated that these scaffolds, microfabricated to mimic cardiac muscle mechanical properties, promote seeded heart cell alignment .
Engineered cardiac tissue (ECT) has also been generated by seeding mouse embryonic stem cell-derived cardiomyocytes into collagen type I supplemented with Matrigel . After in vitro stretching for 7 days, authors demonstrated that the ECT can beat synchronously and respond to physical and pharmaceutical stimulation. In addition, no signs of tumorigenesis were found after subcutaneous implantation for 4 weeks. Cultivation of mESC-derived cardiomyocytes on elastic poly(lactide-co-caprolactone) scaffolds exposed to cyclic stretch, followed by implantation into infarcted hearts showed reduced fibrotic tissue formation and upregulation of cardiac gene expression as compared with unstrained controls . Cyclic stretch was also described to upregulate expression of sarcomeric cardiac genes and to improve cell alignment and distribution of connexin-43, a gap junction protein, highlighting the importance of mechanical strain transduction in cardiac tissue engineering .
A different, complementary approach consists in the preservation of cardiac tissue by rescuing the vascular network compromised during myocardial infarction. Many authors have now described the potential to generate vascular-related cells such as endothelial and perivascular cells from hESCs [40–42]. Injection of hESC-derived vascular cells in a bioactive hydrogel as an in situ forming scaffold after myocardial infarction in rats has shown that the delivered cells formed capillaries in the infarct zone . In addition, magnetic resonance imaging revealed that the microvascular grafts effectively preserved contractile performance, attenuated left ventricular dilation, and decreased infarct size .
In a more complex approach, Caspi et al.  have engineered vascularized cardiac muscle using hESC-derived cardiomyocytes and hESC-derived endothelial cells seeded in biodegradable, biocompatible, and Food and Drug Administration (FDA) approved materials. Analysis of the engineered tissues indicated that increased cardiomyocyte and endothelial cell proliferation as well as formation of vessel-like structures occurred when these cells were cultured with mouse embryonic fibroblasts. When implanted into an uninjured rat heart, these tri-culture scaffolds displayed the formation of viable grafts with both human and host-derived patent vasculature within the implants . A similar tri-culture approach was utilized by Stevens et al.  who reported that patches containing only cardiomyocytes do not form substantial grafts in vivo demonstrating the importance of both vascular and stromal elements in increasing survival and integration of the engineered cardiac tissue. However, neither group assessed the presence of residual undifferentiated cell activity in the implanted engineered tissues.
A promising cell type for use in cardiac tissue-engineering strategies would be iPS cells due to their documented ability to give rise to functional cardiomyocytes . The use of these cells would not only overcome the ethical concerns related to the use of hESCs, but it might also allow for the generation of an unlimited supply of functional, proliferative, and possibly autologous human cardiomyocytes and vascular cells thus overcoming any immunogenic concerns as well. However, iPS cells hold the similar safety issues involving differentiation into non-cardiac cell types or teratomas.
Adult Stem Cells
While there are still ethical and immunogenic concerns related to the use of hESCs in tissue engineering with therapeutic purposes, the use of autologous adult cells as a source overcomes those issues. Adult stem cells with the potential to self-renew and to differentiate into specific lineages exist in different organs. Some examples include hematopoietic and mesenchymal cells of bone marrow. Some reports have implicated adult bone marrow cells in myocardial regeneration [47–49] as well as functional improvement in infarcted hearts . The current data indicate that bone marrow cells improve cardiac function by a paracrine mechanism dependent on the secretion of soluble factors and not trans-differentiation . Nonetheless, the contradictory results obtained with bone marrow transplantation in patients with infarcted myocardium  and issues regarding the possible formation of bone and cartilage from these cells still need to be addressed before these cells can be safely used for cardiac therapy.
Another possible source of cells for cardiac engineering is resident cardiac stem cells. Cardiac progenitor cells expressing stem cell antigen-1 (Sca-1) have been reported in adult mouse myocardium  and have been implicated in cardiac homing and differentiation after infarction. Others report the existence of Lin(−) c-kit(+) cells that, when injected into an ischemic heart, reconstitute well-differentiated myocardium  and resident Isl1+ cardiac progenitors that have been shown to hold the potential to differentiate into cardiomyocytes . However, their real potential for cardiovascular tissue engineering is still unknown given the fact that these cells are present in very low numbers.
Towards this goal, Domian et al.  employed a two-colored fluorescent reporter system (eGFP driven by a cardiac-specific Nkx-2.5 enhancer and dsRed driven by an Isl1-specific enhancer of the Mef2c gene) to isolate first and second heart field progenitors from mouse embryos and embryonic stem cells and to generate beating two-dimensional cardiac tissue. Different populations were isolated according with their expression profile of the above-mentioned fluorescent reporters by fluorescence-activated cell sorting. Authors show that the eGFP+/dsRed + Isl1 population was most similar to the myogenic population. When seeded on glass surfaces coated with poly(dimethyl siloxane) stamped with micropatterned fibronectin lanes, the double positive cells elongated in the direction of the patterns, expressed sarcomeric alpha-actinin, and formed muscular thin films (MTF) which generated contractile force comparable with that of neonatal rat ventricular cardiomyocytes . Importantly, the MTF displayed spontaneous beating and could also be paced by field stimulation.
Adult cardiac progenitors can also be isolated from explant cultures of human endomyocardial biopsies and expanded in vitro as self-adherent clusters or cardiospheres (CSps) [56, 57]. These cells displayed both paracrine and direct regeneration effects in infarcted mice . They were shown to secrete vascular endothelial growth factor, hepatocyte growth factor and insulin-like growth factor-1 when transplanted into a mouse model of myocardial infarction accounting for a paracrine activity that decreased apoptotic rates and caspase 3 levels while increasing capillary density . In addition, based on the number of human-specific cells relative to overall capillary density and myocardial viability, direct differentiation accounted for 20% to 50% of the observed effects. The same group also described that magnetic targeting of iron-labeled cardiosphere-derived cells enhanced engraftment and functional benefits . In order to do that, CSps-derived cells were labeled with superparamagnetic microspheres and guided to the heart by imposing an external magnetic field on the heart during and immediately after cell injection. With this approach, injected cells accumulated around the ischemic zone while the non-targeted cells washed out immediately after injection. Quantitative polymerase chain reaction analysis confirmed cell retention (24 h post-delivery) and engraftment (3 weeks post-delivery) in the recipient hearts by threefold compared with non-targeted cells . Maximal attenuation of left ventricular remodeling and greatest functional improvement occurred in the cell-targeted group without incremental inflammation .
Challenges and Future Studies
Embryonic and induced pluripotent cell-based therapies are still in their infancy with respect to clinical translation. To date, only one embryonic stem cell-based therapy for spinal cord injury has been approved for clinical trial . The use of embryonic stem cells for cardiac regeneration is currently in preclinical animal trials , although there have been several other cell-based therapies in clinical trials involving adult-derived progenitor cells or stem cells for cardiac repair [61–68]. The risks associated with embryonic stem cell-derived therapies come from the possibility that even one undifferentiated cell, if present, could potentially result in the formation of teratoma. While researchers continue to elucidate the mechanisms of novel therapies that have shown great promise in animal studies, clinicians are more concerned with the unmet clinical needs of their patients .
The success of a potential therapy for cardiac regeneration not only depends on the ability to provide the necessary physiological improvements but also on the ability to achieve clinical implementation. Seger and Lee  outlined a few considerations that should be carefully examined when investigating a potential stem cell-based therapy for cardiac regeneration. First and foremost come the issues of cell source and isolation. While ESC can reliably give rise to large numbers of cardiomyocytes, potential immunogenicity of differentiated progeny and safety issues related to the residual undifferentiated cell activity remain. iPSC represent a potential autologous cell source, however, the time required for reprogramming and differentiation into cardiomyocytes, coupled with the low cardiomyocyte yield of current protocols  represent significant challenges for potential clinical translation. Viral incorporation of reprogramming genes into the host genome and the complexity of epigenetic modifications involved in reprogramming represent another challenge [69–71].
Most cell injection studies have reported significant drawbacks with respect to the cell retention, viability, and distribution [26, 72] of the injected cells. Recent advances in tissue-engineered constructs have shown great promise in providing the necessary support for cell retention and distribution [73–75]. However, issues related to the survival of cells in thick constructs remain. Functional coupling with the host myocardium is yet to be demonstrated for cardiac tissues based on human pluripotent stem cell-derived cardiomyocytes. The work of Zimmermann and Eschenhagen in the rat system clearly demonstrated the functional integration of the engineered tissue with the host myocardium . In addition, although direct cell/biomaterial injection can be performed in a minimally invasive catheter-based route, implantation of engineered cardiac tissues requires open heart surgery thus it would be limited to a small number of patients. Issues related to storage and transport of these human-engineered cardiac tissues would also emerge and affect clinical availability. The tissue engineering bioreactor would likely assume the roles of the cultivation, storage, and transport vessel, thus motivating further progress in the bioprocess and bioreactor field.
Safety is perhaps the most important consideration when translating this technology to the clinics. If pluripotent stem cells are to be used in future therapies, rigorous purification of desired cell populations are required. New in vitro assays that test the residual undifferentiated cell activity and the stability of the differentiated progeny are required. Towards this goal, we have demonstrated that the in vitro engineered heart tissue can be a useful model for cell injection studies . We have evaluated the regenerative potential of injected pluripotent stem cell-derived cardiomyocytes (ESC-CMs) as well as pluripotent stem-cell-derived Flk1+/PDGFα + cardiac progenitors (ESC-CPs). We used EHTs as surrogate heart tissues and studied their ability to integrate with injected ESC-CMs, ESC-CPs, neonatal cardiomyocytes, or neonatal cardiac fibroblasts. Functional and phenotypic analyses (Fig. 2) revealed that ESC-CPs improved excitation threshold, maximum capture rate, impulse propagation, and expression of cardiac markers such as cardiac troponin T and connexin while the other cell types exhibited limited to no functional integration with the surrogate EHT . Additionally, EHT was instrumental in enabling identification of residual undifferentiated cell activity in mouse ESC-derived populations . Injection of undifferentiated R1 mouse ESC into EHT leads to teratoma formation and formation of structures characteristics of all three germ layers consistent with the in vivo studies . The long-term cultivation (4 weeks) of Flk1+/PDGFα + cardiac progenitors in the absence of cardiac inductive cytokines resulted in the formation of non-cardiac mesodermal structures such as bone and cartilage, reinforcing the importance of high purity selection of differentiated progeny and maintenance of cardiac inductive cytokines. By consequence, studies using mouse ESCs also represent tools to investigate the importance of biomaterials and the influence of different scaffolds in the assembly of cardiac tissue in vitro.
We have also developed a cardiac tissue based on neonatal rat heart cells that mimics some aspects of diabetic myocardium, by modulation of glucose and insulin concentrations during cultivation. Our results indicate that the diabetic rat heart and high glucose cultivation conditions exhibited diminishing electrophysiological properties and increased ratio of myosin heavy chain isoforms β to α, indicative of diseased states . Our current studies involve miniaturization of the EHT for high-throughput studies and development of disease specific models.
While it is clear that progress has been made in cardiac tissue engineering and regenerative medicine, additional investigation is required in order to reach a consensus on the best biomaterials and cell types to be used as well as the optimal time point for cell injection or tissue implantation. This would be facilitated by the standardization of experimental conditions and would require the systematic analysis of many cell types and biomaterials. From the studies described above, we can conclude that the most important consideration to be addressed by new therapeutic strategies is long-term benefit that includes stable functional improvements and attenuation of pathological remodeling. For these goals to be achieved, efficient delivery and survival of the implanted cells and engineered tissues is required. Since human post-natal cardiomyocytes have limited ability to proliferate, the current lack of an autologous non-immunogenic source of cardiomyocyte has limited the progress of cardiac tissue engineering towards clinical applications. Functional cardiac tissues have been engineered based on hESC derived cardiomyocytes and the lessons learned from these studies pave the way to tissue engineering of cardiac patches based on human iPSC.
Financial support for our work is provided by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN 326982–10), Discovery Accelerator Supplement (RGPAS 396125–10), NSERC Strategic Grant (STPGP 381002–09), NSERC-Canadian Institutes of Health Research Collaborative Health Research Grant (CHRPJ 385981–10), and Heart and Stroke Foundation of Ontario Grant-in-Aid (T6946).