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ESC-based heart therapy
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 9, No. 4 2012
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Induced pluripotent stem cells
Preclinical animal models for testing iPSC/ESC-based heart therapy Francesca Stillitano1, Ioannis Karakikes1, Kevin D. Costa1, Kenneth Fish1, Roger J. Hajjar1, Jean-Se´bastien Hulot1,2,* 1 2
Cardiovascular Research Center, Mount Sinai School of Medicine, New York, USA UPMC Univ Paris 06, UMR_S 956, Paris, France
Embryonic and induced pluripotent stem cell (ESC/
Section editor: Ronald Li – LKS Faculty of Medicine, University of Hong Kong, Hong Kong, and Mount Sinai School of Medicine, New York, NY, USA.
iPSC) technologies offer an unprecedented possibility of devising cell replacement therapies for numerous disorders, including cardiovascular diseases. Studies are progressively investigating whether ESC or iPSC can serve to restore physiological function of diseased hearts in vivo. However, the animal model, the population of ESC/iPSC-derived cardiomyocytes and the delivery technique need to be optimized before these cells can be used effectively for cell replacement therapy. In this review, we describe the potential applications, limitations and challenges of iPSC- and ESCbased heart therapies in preclinical animal models.
Introduction Pluripotent stem cells have the ability to differentiate into cell types of all three germ layers, including cardiac and vascular cells [1–3]. Human embryonic stem cells (ESCs), which are derived from the inner cell mass of blastocyst stage embryos, have the unique ability to self-renew indefinitely while maintaining the potential to give rise to all cell types in the human body [4]. The revolutionary discovery of induced pluripotent stem cells (iPSCs), whereby a patient’s somatic cells can be reprogrammed into an embryonic pluripotent state by the forced expression of a defined set of transcription factors [5], has provided another source of stem cells enabling *Corresponding author.: J.-S. Hulot ([email protected]) 1740-6757/$ ß 2012 Published by Elsevier Ltd.
in vitro disease modeling and drug testing, and in vivo cell replacement therapies [6]. The heart is one of the least regenerative organs in the body and consequently, loss of cardiomyocytes after injury (i.e., infarction or other diseases) often leads to heart failure. Hence, a large number of mature ventricular myocytes are needed for replacement therapy. However, human donor hearts and cardiomyocytes are in extremely limited supply, motivating a demand for alternative cardiomyocyte sources. Moreover, in contrast to cardiomyocytes, the use of less mature stem cell-derived progenies (i.e., cardiovascular progenitors) might not only restore myocardial tissue but could also contribute to revascularization and augment contractility via direct or indirect cell–cell interactions. Different types of autologous cells (including skeletal myoblasts, hematopoietic stem cells and mesenchymal stem cells) have been tested in preclinical and early-stage clinical trials but with inconsistent results [7–9]. It is now widely agreed that these cells do not form a significant amount of new myocardium. Instead, their potential benefit is linked to nonmyogenic mechanisms such as reduced ventricular dilation or paracrine activation of cytoprotective signaling pathways [10,11]. The seminal achievement of pluripotency holds great promise for regenerative medicine and the use of ESC or iPSC as a cell source for cardiac repair has thus become an emerging and exciting field.
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Smooth muscle
Ectoderm (SOX1, PAX6)
ES/iPS cells
Oct4 NANOG SOX2
Primitive streak mesoderm
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Brachyury T MIXL-1
MESP1,2 Flk-1
Cardiac progenitors cells
Early Cardiomyocytes
NKX2.5 GATA4 Tbx5 Mef2C
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cTnT αActin MLC2a MLC2v SCN5A CACNA IRX4
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Specific Wnt/β-catenin pathway antagonists
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Differentiation Days Drug Discovery Today: Disease Models
Figure 1. Schematic representation of cardiac differentiation from pluripotent stem cells.Cardiac differentiation process starts by the introduction of exogenous factors (TGFb superfamily proteins) which induce the primitive streak mesoderm. The processes which follow this step are still not clear. However, the later introduction of a Wnt signal inhibitor will push the cells further to form cardiomyocytes. Each step of differentiation is represented by the expression of specific transcription factors.
Although mouse ESC were first isolated in 1981 and human ESC have been available for more than a decade [4], studies involving their transplantation into the heart have begun only recently. A series of studies have recently demonstrated that iPSC form functional cardiovascular cell lineages with similar efficiency as ESCs in vitro. Overall, only a limited number of studies have investigated whether ESC or iPSC can serve to restore physiological function of diseased hearts in vivo. One of the reasons is linked to the ability of undifferentiated embryonic stem cells to form teratomas, an encapsulated tumor that contains tissue components resembling normal derivatives of all three germ layers, when transplanted into normal or diseased hearts. Similar concerns have been raised with IPS cells. As a consequence and in contrast to other cell types used for therapies, transplantation of ESC or iPSC will require some degree of predifferentiation to restrict the cells’ repertoire and prevent tumor growth (Fig. 1). Different protocols are progressively proposed to achieve an efficient cardiogenesis of ESC and/or iPSC and thus support their use in in vivo models. e230
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At this time, the in vivo testing of iPSC- and ESC-based heart therapies remains limited and substantial challenges must be addressed and resolved before successful translation to human patients. As described below, the key issues include: 1. Defining the optimal animal model, 2. Identifying the optimal stem cells to be delivered, 3. Determining the optimal stem cell delivery route to the myocardium, 4. Defining the appropriate endpoints post-delivery.
Defining the optimal animal model Table 1 summarizes selected ESC and iPSC heart transplantation studies in animals conducted to date. Initial studies were conducted in naı¨ve animals and have demonstrated the capacity of ESC-derived cardiomyocytes to form new myocardium in uninjured hearts. In these studies, embryoid body-based differentiation was performed and then enriched for cardiomyocytes either by physical dissection of beating areas [12,13] or by Percoll gradient centrifugation [14]. The cardiomyocyte purity was about 60–70% with the majority of remaining cells exhibiting an epithelial phenotype. Interestingly, after
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Drug Discovery Today: Disease Models | Induced pluripotent stem cells
Table 1. Summary of selected ESC and iPSC implantation studies Referencea
Type of stem cell
Animal model
Delivery
Maximal follow-up
Heart function
Laflamme, 2007 [19]
Human ESC-derived CM + prosurvival cocktail
- Athymic nude rats - I-R injury (60 min)
Direct injection 4 days after I/R
28 days
Significant improvement of contractile function in ESC treated rats
Dai, 2007 [18]
Human ESC-derived CM
- Athymic nude rats - I-R injury (15 min)
Direct injection 5 min after coronary occlusion
28 days
N/A
Kolossov, 2006 [16]
Mouse ESC-derived CM
Syngeneic mice MI by LCA ligation or cryolesion
Direct injection at the time of surgery
28 days
Significant improvement of contractile function in ESC treated animals
Van Laake, 2007 [17]
Human ESC-derived CM
SCID mice MI by LCA ligation
Direct injection at the time of surgery
12 weeks
Transient improvement of contractile function at week 4 in ESC treated mice; no differences at week 12
Caspi, 2007 [15]
Human ESC-derived CM
SD rats MI by LCA ligation
Direct injection 7–10 days after surgery
60 days
Significant improvement of contractile function in ESC treated rats
Bel, 2010 [48]
Primate ES-derived CM
Rhesus monkeys I-R injury (120 min)
Composite cell sheet applied 2 weeks after surgery
4 weeks
Non-significant for improvement; no teratoma formation
Mauritz, 2011 [54]
Mouse iPSC-derived cardiovascular progenitors
SCID mice MI by LCA ligation
Direct injection at the time of surgery
14 days
Significant improvement of contractile function in iPSC treated animals
a First author, year. Embryonic stem cell (ES), induced pluripotent stem cell (iPSC), cardiomyocyte (CM), ischemia-reperfusion (I-R), myocardial infarction (MI), left coronary artery (LCA), Sprague Dawley (SD), severe combined immunodeficiency (SCID).
transplantation into naı¨ve athymic (nude) rat myocardium, there was a progressive enrichment of cardiomyocytes and loss of the epithelium. Human cardiomyocyte grafts volume increased seven-fold over a 4-week period [14] because of robust proliferative capacity both in vitro and in vivo [14,15]. Moreover, unlike skeletal myoblast-grafts, the ESC-derived cardiomyocytes were able to achieve some electrical integration with surrounding host myocardium [12]. All of the transplantation studies in pathological hearts have been conducted in the context of ischemic injury. Most of the studies were conducted in rodent models of myocardial infarction (MI) following permanent [15–17] or temporary (ischemia/reperfusion infarct) [18,19] coronary artery ligation. This injury model is clinically relevant because ischemic cardiomyopathy is the leading cause of death among people in industrialized nations and the leading cause of heart failure. By contrast, the infarcted heart (especially at an early stage) is a harsh environment for the transplantation of cells because of reduced blood flow in the infarcted area, the increased ischemic injury in the remote area, the progressive loss of matrix integrity, the abnormal tissue mechanical properties, and the cellular inflammatory response. As a consequence, the engraftment success rate is significantly lower in infarcted than in normal hearts [19,20].
Even if significant differences exist between protocols (mainly animal models, timing of injection, differentiation protocols) as shown in Table 1, all of these studies were able to demonstrate the presence of stable cell grafts within the infarcted tissue. The grafted ESC-CMs or iPSC-CMs were mainly found as confluent cell clusters in the infarct border zone. Functional studies showed that transplantation of ESCor iPSC-CMs improved the left ventricular contractile function or at least prevented its worsening as compared to nontreated animals. This improvement was however noted on short-term evaluation of cardiac function (i.e., 1 month after ESC- or iPSC-CMs administration) but was transient in one study that also performed 3-month follow-ups. This result was reproduced in an independent experiment using a higher amount of injected cells [21] thus raising concerns about the long-term impact of ESC-CMs administration. Interestingly, there was a manifest graft survival without reduction in graft size up to three months after ESC-CM administration. Van Laake et al. found that 80–95% of the surviving donor cells had a cardiomyocyte phenotype thus raising concerns whether the generated cardiomyocytes are adhering to and in communication with the surrounding myocardium and are thus able to contribute to the systolic force generation. The development of electromechanical junctions between www.drugdiscoverytoday.com
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the grafted cells and the surrounding host cardiomyocytes is required for synchronous contraction. Expression of connexin 43, a marker of electrical coupling normally found in gap junctions between cardiomyocytes, has been reported in some grafted cells but with limited formation of mature junctional structures. Similarly, the organization of desmosomes (which provide mechanical continuity between cardiomyocytes) in grafted cells is largely unknown [17]. The choice of the animal model might impact the issue of cell– cell interaction [12]. It is likely that human ESC- or iPSC-CM fail to couple to rodent host myocardium because human cells cannot keep up with high heart rate of a rodent. The use of large-animal models for testing human-derived cardiac stem cell therapies should thus be preferable, but appears to be very limited so far. In an elegant study, Blin et al. administered Rhesus ESC-derived cardiovascular progenitors (selected on the SSEA-1 cell surface marker) into a Rhesus monkey model of MI [22] created by a 90-min coronary occlusion/reperfusion protocol; the selected cells were injected 2 weeks later in the infarcted area during an openchest surgery. These cells engrafted into the infarcted monkey hearts and differentiated into morphologically mature cardiomyocytes. Other large-animal preclinical models could be proposed to study cardiac stem cell therapies, especially pigs which have well-documented morphological and functional similarities to humans. Larger animals are also more compatible with minimally invasive catheter-based techniques for inducing MI [23] thus avoiding many unwanted complications of open-chest surgery. While isolation of ESC have not yet been achieved in these animals, recent studies have shown that porcine somatic cells (i.e., skin fibroblasts) can be reprogrammed into pig-iPS cells using transfer of the standard Yamanaka reprogrammation factors [24]. The generation of pig-iPSC and their potential to further differentiate into the three embryonic germ layers open new possibilities for the preclinical application of iPSCs. From a theoretical point of view, iPSC technology offers the opportunity to perform autologous transplantation, thus circumventing the rejection issues seen with xeno- or allo-transplantation. To ensure the survival of grafted cells coming from other species or from other animals, investigators often use immunosuppressive agents (mainly cyclosporine A) in immunocompetent animals. It has recently been shown that cyclosporine inhibits the opening of mitochondrial permeability-transition pores and thereby limits myocardial injury that occurs after ischemia-reperfusion [25], possibly impacting the conclusions drawn from such studies. Alternatively, researchers have used immunodeficient animals (such as SCID mice), which restricts investigation to small animal models. Therefore, the expanded development and optimization of large-animal preclinical models in which autologous iPSC-derived cells can be used for implantation is considered a critical step e232
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toward the successful translation of stem cell therapies for patients in need of cardiac repair.
Identifying the optimal stem cells to be delivered As previously mentioned, it is critical that ES or iPS cellderived preparations intended for in vivo transplantation require some degree of pre-delivery differentiation to avoid the formation of tumors arising from undifferentiated pluripotent stem cells (Fig. 1). Teratoma formation in treated hearts could cause life-threatening events such as fatal arrhythmias, cardiac tamponnade, and heart failure. Therefore, given the heterogeneous cell mixture within embryoid bodies (EBs), purification of cardiac cells is crucial not only to eliminate remaining undifferentiated cells, but also to derive a pure population of differentiated cardiomyocytes. Although EB dissection and centrifugation sorting techniques have been used to enrich cardiomyocyte selection [26,27], the achieved purity is probably insufficient for clinical purposes. Alternatively, the generation of transgenic ESC allows the identification and selection of differentiating cardiomyocytes. This approach uses a cardiac-specific promoter to drive expression of a selectable marker or reporter gene. For example, the ventricular human myosin light chain 2v (MLC2v) promoter can be used to create a stable transgenic hESC lines in which eGFP-expressing cells, appearing during in vitro differentiation, can be identified and sorted [28]. A similar strategy can be used to specifically enrich atrial versus ventricular myocytes [29]; however, genetic modification is required. To overcome this limitation, a fluorescent dye that labels mitochondria has been used to selectively mark ES/iPS cell-derived cardiomyocytes [30], yielding highly pure cardiomyocytes (99% purity) obtained by fluorescence-activated cell sorting. Such nongenetic methods can be powerful tools in the clinical setting. Several studies found no teratomas in hearts transplanted with cardiomyocytes or cardiac progenitors derived from purified ES cells [19,31], suggesting the risk of tumor formation might be low after appropriate cell sorting. However, further analysis of the therapeutic potential of ES/iPS cells and their progeny, including the risk of teratoma-forming propensity in animal models with long-term follow-up, is required before cell therapy with human iPS cells can advance to clinical trials. Because ESCs are not of patient origin, the immune response problem must be addressed. Transplant rejection occurs due to allelic differences in the surface antigens expressed by donor and recipient. There are three distinct types of transplantation antigens: ABO blood group antigens, minor histocompatibility antigens and major histocompatibility complex (MHC) molecules. All three are expected to be found in cell populations used to create cardiac grafts. However, allelic differences in MHC molecules are, by far, the most significant immunological barrier to organ transplantation, including ESC-derived cell transplants. Undifferentiated
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hESCs are sometimes called ‘immunoprivileged’ because they express low levels of class I MHC (MHC-I) molecules [32–34]. Unfortunately, MHC-I expression increases 10-fold with the induction of differentiation [34]. Thus, stem cell graft rejection may be elicited when the recipients’ allogeneic T cells recognize the foreign MHC-I surface molecules, whether differentiation occurs before or after transplantation. Several methods have been proposed to address immunorejection [35]. Among them, derivation of ESCs that have their MHC [also called human leukocyte antigen (HLA) in humans] matched with the patient’s could be a potential method for minimizing immunorejection in ESC transplantation [36,37]. Moreover, the development of iPSC-derived differentiated cells has been expected to provide personalized sources for cell-based therapy. iPS cells were originally believed to be non immunogenic. However, recent data have raise concerns about the potential immunogenicity of these cells. A recent study reported that the transplantation of immature iPSCs induced a T-cell-dependent immune response even in a syngeneic mouse, likely due to abnormal gene expression [38]. This immunogenicity could be linked to the over-expression of minor antigens [38], potentially resulting from punctual mutations in the coding sequences of iPSCs [39] or the epigenetic regulation of iPSCs [40]. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPS cells must be carefully evaluated before clinical application into human patients. The use of ESC/iPSC-derived cardiomyocytes for basic developmental research and large-scale applications, such as largeanimal preclinical studies, has been hampered by their poor yield from typically heterogeneous stem cell cultures. This challenge was in part solved by studies that carefully tested signaling molecules and intracellular mediators that are known to drive cells in the earliest steps of cardiac development under normal conditions (Fig. 1). Some small molecules have the potential to enhance the differentiation from ES cells to specific tissue cells. A high-throughput screening system has been used to identify such small molecules [41,42], revealing that ascorbic acid [43], activin A and bone morphogenetic protein-4 [31] enhance the differentiation of ESCs into cardiomyocytes. Thus, by optimizing growth factor combinations and timing, cardiac myocyte differentiation rate can be increased and, importantly, the new protocols can be applied to essentially all pluripotent stem cell lines including iPS cells [44,45].
around the infarcted myocardium. Cell retention and survival is consistently low when injected into the myocardium, and even lower with intracoronary infusion [19,46,47]. Therefore, alternative methods, including epicardial deposition of cellularized biomaterial, have been recently proposed. The underlying concept is to provide an external supply of cardiac-specified stem cells through a three-dimensional (3D) construct made of cardiac progenitors and feeder cells placed on a composite cell sheet. The cell sheet is then applied on the infarcted area during cardiac surgery. In a non-human primate model, it has been shown using this technique that progenitor cells migrated to the myocardium with a robust engraftment [48]. The development of substrate-mediated cell therapy which takes advantage of the ability of biomaterial sheets to act as a flexible patch, while enabling direct apposition of exogenously introduced cells, has also been reported [49]. A recent percutaneous technique for gelfoamenabled pericardial delivery of cells to the infarcted myocardium offers another promising approach for stem cell therapies [50]. Finally, there is little evidence to support the intuitively attractive idea that the adult mammalian heart provides a ‘cardiogenic environment’ that will drive maturation and orientation of cardiac myocytes from pluripotent stem cells. An alternative approach to address this issue relies on tissue engineering of cardiac muscle from human ESCs and iPSCs. In two recent publications [51,52], it has been shown that the ex vivo combination of ESC/iPSC with polymeric scaffolds allows the generation of tissue-engineered muscle constructs. The engineered tissues show coherent contractions 5–10 days after casting. They display a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes, suggesting that the 3D tissue format improves the maturation of cardiomyocytes, although they remained genetically similar to EBs [51]. It has been suggested that a geometrically straight structure of the tissue and cyclic strain favor cardiomyocytes maturation [51]. In one study, human bioengineered cardiac tissues was transplanted onto the epicardium of a healthy athymic rat heart, showing graft survival and connection to the host myocardium and coronary circulation [52]. These data suggest that in vivo engraftment of engineered cardiac tissue created from ESC- or iPSC-derived cardiomyocytes represents an interesting alternative strategy to direct cell injection therapy that warrants further investigation.
Determining the optimal stem cells delivery route to the myocardium
Defining the appropriate endpoints post-delivery
Cell viability and integration into the host anatomical structure is an additional challenge to stem cell therapy for cardiac repair. In most studies, ESC- and iPSC-based therapies have been administered through conventional needle-based intramyocardial injections. While the number of cells and injections may vary, the common concept is to deliver cells in or
Finally, there is a need to better define the different endpoints that will be used to provide an overall assessment of ESC/ iPSC-based heart therapies. Most prior studies have qualitatively assessed the presence of engrafted cells in the myocardium of transplanted animals. However the quantitative assessment (i.e., the proportion of cells engrafted/cells transplanted) has proven challenging. Several approaches have www.drugdiscoverytoday.com
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Primitive streak mesoderm
Cardiac committed mesoderm Cardiac progenitors cells Early Cardiomyocytes
Identifyin g the op tima l stem cells to be delivered
Late Cardiomyocytes
ES/iPS cells
Rodents
Large animals
Non human primates
Defining the optimal animal model heart rate ~ 500 bpm
very similar physiology to human, very expensive, animal ethics
physiology and anatomy similar to human (~100 bpmbpm); used for pharmaceutical testing, large to keep, expensive, animal ethics
Determining the optimal stem cells delivery route to the myocardium
needle-based intramyocardial injections Cell sheets, biodegradable polymeric scaffold bioengineered cardiac tissues
Defining the appropriate endpoints post-delivery
Pressure
Ejection
Isovolumetric relaxation
Stroke Volume
Isovolumetric contraction
Filling
PV-Loop (invasive)
MRI non-invasive Drug Discovery Today: Disease Models
Figure 2. Schematic representation of critical steps for testing iPSC/ESC-based heart therapy in preclinical models.
been recently proposed to enable non-invasive stem cell tracking thus helping to follow their migration, homing, division and differentiation after transplantation. MRI cell tracking is particularly appealing in cardiology as it would e234
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allow a simultaneous assessment of cell engraftment and cardiac function. Different approaches including MRI-based reported gene technology [53] have been proposed but their utility in tracking ES/iPS cells fate remain undetermined.
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Other studies have suggested measuring the graft size as a surrogate of cell engraftment [54]. There is a growing consensus that changes in left ventricular ejection fraction might not fully reflect the impact of ESC/iPSC transplantation on cardiac remodeling and cardiac function. Ideally, global cardiac volumes and dimensions as well as cardiac hemodynamic parameters should be augmented with regional measures of myocardial structure and contractile function and assessed at early and late time points after transplantation to more comprehensively evaluate the full impact of cardiac stem cell therapies.
Prospects for the future There are important opportunities to develop an efficient preclinical platform dedicated to the evaluation of stem cell-based heart therapies before application in humans. Figure 2 summarizes the key points that would need to be addressed and proposes directions for future investigations based on current evidence. Porcine models of heart failure are particularly useful for evaluation of novel therapies since they recapitulate clinical presentations of both acute and chronic cardiovascular conditions. Pig-specific iPS cells can be generated from differentiated cells taken in newborns and then reprogrammed to cardiomyocytes. Autologous transplants to pig models of heart failure provide a critical path to clinical application. For example, the evaluation of the iPS-based heart therapy can then be conducted in a model of myocardial ischemia performed on the adult animal from which cells were originally harvested. Alternatively, the creation of a genotyped donor iPS cells library would allow the selection of matched-histocompatible iPS cells to the recipients. In the reprogramming process, in vitro cultures can be engineered to express reporter systems such as the NIS symporter or the HSV-tk system [55], thus enabling longitudinal non-invasive molecular imaging to assess the fate and engraftment rate and fate of administered cells in addition to their impact on cardiac function and remodeling. Conduction velocity studies would help to assess the electromechanical integration of these cells. Further investigations are obviously needed to validate this approach and define the optimal stage cell development, optimal delivery route and time of delivery.
Conclusion The remarkable proliferative and differentiation capacity of stem cells promises an unlimited supply of specific cell types including viable functioning cardiac cells. ESC/iPSC-derived cardiomyocytes represent a suitable cell source that may play a major role in regenerative therapy in the future. However, ES/iPS cell technology has several key issues that remain to be overcome, including the low efficiency of cardiac myocyte differentiation from pluripotent stem cells, the low retention and survival rate of cells when injected or infused into the myocardium, and the possibility of tumor formation in vivo.
Drug Discovery Today: Disease Models | Induced pluripotent stem cells
Solving these issues in suitable large-animal preclinical models will be essential for successfully translating ESC/iPSC technology to improve the health of millions of human patients suffering from heart disease.
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Stadtfeld, M. et al. (2010) Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465, 175–181 Borowiak, M. et al. (2009) Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell 4, 348–358 Chen, S. et al. (2009) A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat. Chem. Biol. 5, 258–265 Takahashi, T. et al. (2003) Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 107, 1912–1916 Kattman, S.J. et al. (2011) Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 Burridge, P.W. et al. (2011) A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6, e18293 Qiao, H. et al. (2009) Death and proliferation time course of stem cells transplanted in the myocardium. Mol. Imaging Biol. 11, 408–414 Zhang, M. et al. (2001) Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J. Mol. Cell. Cardiol. 33, 907–921 Bel, A. et al. (2010) Composite cell sheets: a further step toward safe and effective myocardial regeneration by cardiac progenitors derived from embryonic stem cells. Circulation 122 (Suppl.), S118–S123 Chen, Q.Z. et al. (2010) An elastomeric patch derived from poly(glycerol sebacate) for delivery of embryonic stem cells to the heart. Biomaterials 31, 3885–3893 Ladage, D. et al. (2011) Delivery of gelfoam-enabled cells and vectors into the pericardial space using a percutaneous approach in a porcine model. Gene Ther. 18, 979–985 Schaaf, S. et al. (2011) Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS One 6, e26397 Tulloch, N.L. et al. (2011) Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ. Res. 109, 47–59 Campan, M. et al. (2011) Ferritin as a reporter gene for in vivo tracking of stem cells by 1.5-T cardiac MRI in a rat model of myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 300, H2238–H2250 Mauritz, C. et al. (2011) Induced pluripotent stem cell (iPSC)-derived Flk-1 progenitor cells engraft, differentiate, and improve heart function in a mouse model of acute myocardial infarction. Eur. Heart J. 32, 2634–2641 Zhang, W.Y. et al. (2011) Imaging cardiac stem cell therapy: translations to human clinical studies. J. Cardiovasc. Transl. Res. 4, 514–522
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 9, No. 4 2012
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Induced pluripotent stem cells
Preclinical animal models for testing iPSC/ESC-based heart therapy Francesca Stillitano1, Ioannis Karakikes1, Kevin D. Costa1, Kenneth Fish1, Roger J. Hajjar1, Jean-Se´bastien Hulot1,2,* 1 2
Cardiovascular Research Center, Mount Sinai School of Medicine, New York, USA UPMC Univ Paris 06, UMR_S 956, Paris, France
Embryonic and induced pluripotent stem cell (ESC/
Section editor: Ronald Li – LKS Faculty of Medicine, University of Hong Kong, Hong Kong, and Mount Sinai School of Medicine, New York, NY, USA.
iPSC) technologies offer an unprecedented possibility of devising cell replacement therapies for numerous disorders, including cardiovascular diseases. Studies are progressively investigating whether ESC or iPSC can serve to restore physiological function of diseased hearts in vivo. However, the animal model, the population of ESC/iPSC-derived cardiomyocytes and the delivery technique need to be optimized before these cells can be used effectively for cell replacement therapy. In this review, we describe the potential applications, limitations and challenges of iPSC- and ESCbased heart therapies in preclinical animal models.
Introduction Pluripotent stem cells have the ability to differentiate into cell types of all three germ layers, including cardiac and vascular cells [1–3]. Human embryonic stem cells (ESCs), which are derived from the inner cell mass of blastocyst stage embryos, have the unique ability to self-renew indefinitely while maintaining the potential to give rise to all cell types in the human body [4]. The revolutionary discovery of induced pluripotent stem cells (iPSCs), whereby a patient’s somatic cells can be reprogrammed into an embryonic pluripotent state by the forced expression of a defined set of transcription factors [5], has provided another source of stem cells enabling *Corresponding author.: J.-S. Hulot ([email protected]) 1740-6757/$ ß 2012 Published by Elsevier Ltd.
in vitro disease modeling and drug testing, and in vivo cell replacement therapies [6]. The heart is one of the least regenerative organs in the body and consequently, loss of cardiomyocytes after injury (i.e., infarction or other diseases) often leads to heart failure. Hence, a large number of mature ventricular myocytes are needed for replacement therapy. However, human donor hearts and cardiomyocytes are in extremely limited supply, motivating a demand for alternative cardiomyocyte sources. Moreover, in contrast to cardiomyocytes, the use of less mature stem cell-derived progenies (i.e., cardiovascular progenitors) might not only restore myocardial tissue but could also contribute to revascularization and augment contractility via direct or indirect cell–cell interactions. Different types of autologous cells (including skeletal myoblasts, hematopoietic stem cells and mesenchymal stem cells) have been tested in preclinical and early-stage clinical trials but with inconsistent results [7–9]. It is now widely agreed that these cells do not form a significant amount of new myocardium. Instead, their potential benefit is linked to nonmyogenic mechanisms such as reduced ventricular dilation or paracrine activation of cytoprotective signaling pathways [10,11]. The seminal achievement of pluripotency holds great promise for regenerative medicine and the use of ESC or iPSC as a cell source for cardiac repair has thus become an emerging and exciting field.
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Differentiation Days Drug Discovery Today: Disease Models
Figure 1. Schematic representation of cardiac differentiation from pluripotent stem cells.Cardiac differentiation process starts by the introduction of exogenous factors (TGFb superfamily proteins) which induce the primitive streak mesoderm. The processes which follow this step are still not clear. However, the later introduction of a Wnt signal inhibitor will push the cells further to form cardiomyocytes. Each step of differentiation is represented by the expression of specific transcription factors.
Although mouse ESC were first isolated in 1981 and human ESC have been available for more than a decade [4], studies involving their transplantation into the heart have begun only recently. A series of studies have recently demonstrated that iPSC form functional cardiovascular cell lineages with similar efficiency as ESCs in vitro. Overall, only a limited number of studies have investigated whether ESC or iPSC can serve to restore physiological function of diseased hearts in vivo. One of the reasons is linked to the ability of undifferentiated embryonic stem cells to form teratomas, an encapsulated tumor that contains tissue components resembling normal derivatives of all three germ layers, when transplanted into normal or diseased hearts. Similar concerns have been raised with IPS cells. As a consequence and in contrast to other cell types used for therapies, transplantation of ESC or iPSC will require some degree of predifferentiation to restrict the cells’ repertoire and prevent tumor growth (Fig. 1). Different protocols are progressively proposed to achieve an efficient cardiogenesis of ESC and/or iPSC and thus support their use in in vivo models. e230
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At this time, the in vivo testing of iPSC- and ESC-based heart therapies remains limited and substantial challenges must be addressed and resolved before successful translation to human patients. As described below, the key issues include: 1. Defining the optimal animal model, 2. Identifying the optimal stem cells to be delivered, 3. Determining the optimal stem cell delivery route to the myocardium, 4. Defining the appropriate endpoints post-delivery.
Defining the optimal animal model Table 1 summarizes selected ESC and iPSC heart transplantation studies in animals conducted to date. Initial studies were conducted in naı¨ve animals and have demonstrated the capacity of ESC-derived cardiomyocytes to form new myocardium in uninjured hearts. In these studies, embryoid body-based differentiation was performed and then enriched for cardiomyocytes either by physical dissection of beating areas [12,13] or by Percoll gradient centrifugation [14]. The cardiomyocyte purity was about 60–70% with the majority of remaining cells exhibiting an epithelial phenotype. Interestingly, after
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Table 1. Summary of selected ESC and iPSC implantation studies Referencea
Type of stem cell
Animal model
Delivery
Maximal follow-up
Heart function
Laflamme, 2007 [19]
Human ESC-derived CM + prosurvival cocktail
- Athymic nude rats - I-R injury (60 min)
Direct injection 4 days after I/R
28 days
Significant improvement of contractile function in ESC treated rats
Dai, 2007 [18]
Human ESC-derived CM
- Athymic nude rats - I-R injury (15 min)
Direct injection 5 min after coronary occlusion
28 days
N/A
Kolossov, 2006 [16]
Mouse ESC-derived CM
Syngeneic mice MI by LCA ligation or cryolesion
Direct injection at the time of surgery
28 days
Significant improvement of contractile function in ESC treated animals
Van Laake, 2007 [17]
Human ESC-derived CM
SCID mice MI by LCA ligation
Direct injection at the time of surgery
12 weeks
Transient improvement of contractile function at week 4 in ESC treated mice; no differences at week 12
Caspi, 2007 [15]
Human ESC-derived CM
SD rats MI by LCA ligation
Direct injection 7–10 days after surgery
60 days
Significant improvement of contractile function in ESC treated rats
Bel, 2010 [48]
Primate ES-derived CM
Rhesus monkeys I-R injury (120 min)
Composite cell sheet applied 2 weeks after surgery
4 weeks
Non-significant for improvement; no teratoma formation
Mauritz, 2011 [54]
Mouse iPSC-derived cardiovascular progenitors
SCID mice MI by LCA ligation
Direct injection at the time of surgery
14 days
Significant improvement of contractile function in iPSC treated animals
a First author, year. Embryonic stem cell (ES), induced pluripotent stem cell (iPSC), cardiomyocyte (CM), ischemia-reperfusion (I-R), myocardial infarction (MI), left coronary artery (LCA), Sprague Dawley (SD), severe combined immunodeficiency (SCID).
transplantation into naı¨ve athymic (nude) rat myocardium, there was a progressive enrichment of cardiomyocytes and loss of the epithelium. Human cardiomyocyte grafts volume increased seven-fold over a 4-week period [14] because of robust proliferative capacity both in vitro and in vivo [14,15]. Moreover, unlike skeletal myoblast-grafts, the ESC-derived cardiomyocytes were able to achieve some electrical integration with surrounding host myocardium [12]. All of the transplantation studies in pathological hearts have been conducted in the context of ischemic injury. Most of the studies were conducted in rodent models of myocardial infarction (MI) following permanent [15–17] or temporary (ischemia/reperfusion infarct) [18,19] coronary artery ligation. This injury model is clinically relevant because ischemic cardiomyopathy is the leading cause of death among people in industrialized nations and the leading cause of heart failure. By contrast, the infarcted heart (especially at an early stage) is a harsh environment for the transplantation of cells because of reduced blood flow in the infarcted area, the increased ischemic injury in the remote area, the progressive loss of matrix integrity, the abnormal tissue mechanical properties, and the cellular inflammatory response. As a consequence, the engraftment success rate is significantly lower in infarcted than in normal hearts [19,20].
Even if significant differences exist between protocols (mainly animal models, timing of injection, differentiation protocols) as shown in Table 1, all of these studies were able to demonstrate the presence of stable cell grafts within the infarcted tissue. The grafted ESC-CMs or iPSC-CMs were mainly found as confluent cell clusters in the infarct border zone. Functional studies showed that transplantation of ESCor iPSC-CMs improved the left ventricular contractile function or at least prevented its worsening as compared to nontreated animals. This improvement was however noted on short-term evaluation of cardiac function (i.e., 1 month after ESC- or iPSC-CMs administration) but was transient in one study that also performed 3-month follow-ups. This result was reproduced in an independent experiment using a higher amount of injected cells [21] thus raising concerns about the long-term impact of ESC-CMs administration. Interestingly, there was a manifest graft survival without reduction in graft size up to three months after ESC-CM administration. Van Laake et al. found that 80–95% of the surviving donor cells had a cardiomyocyte phenotype thus raising concerns whether the generated cardiomyocytes are adhering to and in communication with the surrounding myocardium and are thus able to contribute to the systolic force generation. The development of electromechanical junctions between www.drugdiscoverytoday.com
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the grafted cells and the surrounding host cardiomyocytes is required for synchronous contraction. Expression of connexin 43, a marker of electrical coupling normally found in gap junctions between cardiomyocytes, has been reported in some grafted cells but with limited formation of mature junctional structures. Similarly, the organization of desmosomes (which provide mechanical continuity between cardiomyocytes) in grafted cells is largely unknown [17]. The choice of the animal model might impact the issue of cell– cell interaction [12]. It is likely that human ESC- or iPSC-CM fail to couple to rodent host myocardium because human cells cannot keep up with high heart rate of a rodent. The use of large-animal models for testing human-derived cardiac stem cell therapies should thus be preferable, but appears to be very limited so far. In an elegant study, Blin et al. administered Rhesus ESC-derived cardiovascular progenitors (selected on the SSEA-1 cell surface marker) into a Rhesus monkey model of MI [22] created by a 90-min coronary occlusion/reperfusion protocol; the selected cells were injected 2 weeks later in the infarcted area during an openchest surgery. These cells engrafted into the infarcted monkey hearts and differentiated into morphologically mature cardiomyocytes. Other large-animal preclinical models could be proposed to study cardiac stem cell therapies, especially pigs which have well-documented morphological and functional similarities to humans. Larger animals are also more compatible with minimally invasive catheter-based techniques for inducing MI [23] thus avoiding many unwanted complications of open-chest surgery. While isolation of ESC have not yet been achieved in these animals, recent studies have shown that porcine somatic cells (i.e., skin fibroblasts) can be reprogrammed into pig-iPS cells using transfer of the standard Yamanaka reprogrammation factors [24]. The generation of pig-iPSC and their potential to further differentiate into the three embryonic germ layers open new possibilities for the preclinical application of iPSCs. From a theoretical point of view, iPSC technology offers the opportunity to perform autologous transplantation, thus circumventing the rejection issues seen with xeno- or allo-transplantation. To ensure the survival of grafted cells coming from other species or from other animals, investigators often use immunosuppressive agents (mainly cyclosporine A) in immunocompetent animals. It has recently been shown that cyclosporine inhibits the opening of mitochondrial permeability-transition pores and thereby limits myocardial injury that occurs after ischemia-reperfusion [25], possibly impacting the conclusions drawn from such studies. Alternatively, researchers have used immunodeficient animals (such as SCID mice), which restricts investigation to small animal models. Therefore, the expanded development and optimization of large-animal preclinical models in which autologous iPSC-derived cells can be used for implantation is considered a critical step e232
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toward the successful translation of stem cell therapies for patients in need of cardiac repair.
Identifying the optimal stem cells to be delivered As previously mentioned, it is critical that ES or iPS cellderived preparations intended for in vivo transplantation require some degree of pre-delivery differentiation to avoid the formation of tumors arising from undifferentiated pluripotent stem cells (Fig. 1). Teratoma formation in treated hearts could cause life-threatening events such as fatal arrhythmias, cardiac tamponnade, and heart failure. Therefore, given the heterogeneous cell mixture within embryoid bodies (EBs), purification of cardiac cells is crucial not only to eliminate remaining undifferentiated cells, but also to derive a pure population of differentiated cardiomyocytes. Although EB dissection and centrifugation sorting techniques have been used to enrich cardiomyocyte selection [26,27], the achieved purity is probably insufficient for clinical purposes. Alternatively, the generation of transgenic ESC allows the identification and selection of differentiating cardiomyocytes. This approach uses a cardiac-specific promoter to drive expression of a selectable marker or reporter gene. For example, the ventricular human myosin light chain 2v (MLC2v) promoter can be used to create a stable transgenic hESC lines in which eGFP-expressing cells, appearing during in vitro differentiation, can be identified and sorted [28]. A similar strategy can be used to specifically enrich atrial versus ventricular myocytes [29]; however, genetic modification is required. To overcome this limitation, a fluorescent dye that labels mitochondria has been used to selectively mark ES/iPS cell-derived cardiomyocytes [30], yielding highly pure cardiomyocytes (99% purity) obtained by fluorescence-activated cell sorting. Such nongenetic methods can be powerful tools in the clinical setting. Several studies found no teratomas in hearts transplanted with cardiomyocytes or cardiac progenitors derived from purified ES cells [19,31], suggesting the risk of tumor formation might be low after appropriate cell sorting. However, further analysis of the therapeutic potential of ES/iPS cells and their progeny, including the risk of teratoma-forming propensity in animal models with long-term follow-up, is required before cell therapy with human iPS cells can advance to clinical trials. Because ESCs are not of patient origin, the immune response problem must be addressed. Transplant rejection occurs due to allelic differences in the surface antigens expressed by donor and recipient. There are three distinct types of transplantation antigens: ABO blood group antigens, minor histocompatibility antigens and major histocompatibility complex (MHC) molecules. All three are expected to be found in cell populations used to create cardiac grafts. However, allelic differences in MHC molecules are, by far, the most significant immunological barrier to organ transplantation, including ESC-derived cell transplants. Undifferentiated
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hESCs are sometimes called ‘immunoprivileged’ because they express low levels of class I MHC (MHC-I) molecules [32–34]. Unfortunately, MHC-I expression increases 10-fold with the induction of differentiation [34]. Thus, stem cell graft rejection may be elicited when the recipients’ allogeneic T cells recognize the foreign MHC-I surface molecules, whether differentiation occurs before or after transplantation. Several methods have been proposed to address immunorejection [35]. Among them, derivation of ESCs that have their MHC [also called human leukocyte antigen (HLA) in humans] matched with the patient’s could be a potential method for minimizing immunorejection in ESC transplantation [36,37]. Moreover, the development of iPSC-derived differentiated cells has been expected to provide personalized sources for cell-based therapy. iPS cells were originally believed to be non immunogenic. However, recent data have raise concerns about the potential immunogenicity of these cells. A recent study reported that the transplantation of immature iPSCs induced a T-cell-dependent immune response even in a syngeneic mouse, likely due to abnormal gene expression [38]. This immunogenicity could be linked to the over-expression of minor antigens [38], potentially resulting from punctual mutations in the coding sequences of iPSCs [39] or the epigenetic regulation of iPSCs [40]. Therefore, the immunogenicity of therapeutically valuable cells derived from patient-specific iPS cells must be carefully evaluated before clinical application into human patients. The use of ESC/iPSC-derived cardiomyocytes for basic developmental research and large-scale applications, such as largeanimal preclinical studies, has been hampered by their poor yield from typically heterogeneous stem cell cultures. This challenge was in part solved by studies that carefully tested signaling molecules and intracellular mediators that are known to drive cells in the earliest steps of cardiac development under normal conditions (Fig. 1). Some small molecules have the potential to enhance the differentiation from ES cells to specific tissue cells. A high-throughput screening system has been used to identify such small molecules [41,42], revealing that ascorbic acid [43], activin A and bone morphogenetic protein-4 [31] enhance the differentiation of ESCs into cardiomyocytes. Thus, by optimizing growth factor combinations and timing, cardiac myocyte differentiation rate can be increased and, importantly, the new protocols can be applied to essentially all pluripotent stem cell lines including iPS cells [44,45].
around the infarcted myocardium. Cell retention and survival is consistently low when injected into the myocardium, and even lower with intracoronary infusion [19,46,47]. Therefore, alternative methods, including epicardial deposition of cellularized biomaterial, have been recently proposed. The underlying concept is to provide an external supply of cardiac-specified stem cells through a three-dimensional (3D) construct made of cardiac progenitors and feeder cells placed on a composite cell sheet. The cell sheet is then applied on the infarcted area during cardiac surgery. In a non-human primate model, it has been shown using this technique that progenitor cells migrated to the myocardium with a robust engraftment [48]. The development of substrate-mediated cell therapy which takes advantage of the ability of biomaterial sheets to act as a flexible patch, while enabling direct apposition of exogenously introduced cells, has also been reported [49]. A recent percutaneous technique for gelfoamenabled pericardial delivery of cells to the infarcted myocardium offers another promising approach for stem cell therapies [50]. Finally, there is little evidence to support the intuitively attractive idea that the adult mammalian heart provides a ‘cardiogenic environment’ that will drive maturation and orientation of cardiac myocytes from pluripotent stem cells. An alternative approach to address this issue relies on tissue engineering of cardiac muscle from human ESCs and iPSCs. In two recent publications [51,52], it has been shown that the ex vivo combination of ESC/iPSC with polymeric scaffolds allows the generation of tissue-engineered muscle constructs. The engineered tissues show coherent contractions 5–10 days after casting. They display a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes, suggesting that the 3D tissue format improves the maturation of cardiomyocytes, although they remained genetically similar to EBs [51]. It has been suggested that a geometrically straight structure of the tissue and cyclic strain favor cardiomyocytes maturation [51]. In one study, human bioengineered cardiac tissues was transplanted onto the epicardium of a healthy athymic rat heart, showing graft survival and connection to the host myocardium and coronary circulation [52]. These data suggest that in vivo engraftment of engineered cardiac tissue created from ESC- or iPSC-derived cardiomyocytes represents an interesting alternative strategy to direct cell injection therapy that warrants further investigation.
Determining the optimal stem cells delivery route to the myocardium
Defining the appropriate endpoints post-delivery
Cell viability and integration into the host anatomical structure is an additional challenge to stem cell therapy for cardiac repair. In most studies, ESC- and iPSC-based therapies have been administered through conventional needle-based intramyocardial injections. While the number of cells and injections may vary, the common concept is to deliver cells in or
Finally, there is a need to better define the different endpoints that will be used to provide an overall assessment of ESC/ iPSC-based heart therapies. Most prior studies have qualitatively assessed the presence of engrafted cells in the myocardium of transplanted animals. However the quantitative assessment (i.e., the proportion of cells engrafted/cells transplanted) has proven challenging. Several approaches have www.drugdiscoverytoday.com
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Defining the optimal animal model heart rate ~ 500 bpm
very similar physiology to human, very expensive, animal ethics
physiology and anatomy similar to human (~100 bpmbpm); used for pharmaceutical testing, large to keep, expensive, animal ethics
Determining the optimal stem cells delivery route to the myocardium
needle-based intramyocardial injections Cell sheets, biodegradable polymeric scaffold bioengineered cardiac tissues
Defining the appropriate endpoints post-delivery
Pressure
Ejection
Isovolumetric relaxation
Stroke Volume
Isovolumetric contraction
Filling
PV-Loop (invasive)
MRI non-invasive Drug Discovery Today: Disease Models
Figure 2. Schematic representation of critical steps for testing iPSC/ESC-based heart therapy in preclinical models.
been recently proposed to enable non-invasive stem cell tracking thus helping to follow their migration, homing, division and differentiation after transplantation. MRI cell tracking is particularly appealing in cardiology as it would e234
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allow a simultaneous assessment of cell engraftment and cardiac function. Different approaches including MRI-based reported gene technology [53] have been proposed but their utility in tracking ES/iPS cells fate remain undetermined.
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Other studies have suggested measuring the graft size as a surrogate of cell engraftment [54]. There is a growing consensus that changes in left ventricular ejection fraction might not fully reflect the impact of ESC/iPSC transplantation on cardiac remodeling and cardiac function. Ideally, global cardiac volumes and dimensions as well as cardiac hemodynamic parameters should be augmented with regional measures of myocardial structure and contractile function and assessed at early and late time points after transplantation to more comprehensively evaluate the full impact of cardiac stem cell therapies.
Prospects for the future There are important opportunities to develop an efficient preclinical platform dedicated to the evaluation of stem cell-based heart therapies before application in humans. Figure 2 summarizes the key points that would need to be addressed and proposes directions for future investigations based on current evidence. Porcine models of heart failure are particularly useful for evaluation of novel therapies since they recapitulate clinical presentations of both acute and chronic cardiovascular conditions. Pig-specific iPS cells can be generated from differentiated cells taken in newborns and then reprogrammed to cardiomyocytes. Autologous transplants to pig models of heart failure provide a critical path to clinical application. For example, the evaluation of the iPS-based heart therapy can then be conducted in a model of myocardial ischemia performed on the adult animal from which cells were originally harvested. Alternatively, the creation of a genotyped donor iPS cells library would allow the selection of matched-histocompatible iPS cells to the recipients. In the reprogramming process, in vitro cultures can be engineered to express reporter systems such as the NIS symporter or the HSV-tk system [55], thus enabling longitudinal non-invasive molecular imaging to assess the fate and engraftment rate and fate of administered cells in addition to their impact on cardiac function and remodeling. Conduction velocity studies would help to assess the electromechanical integration of these cells. Further investigations are obviously needed to validate this approach and define the optimal stage cell development, optimal delivery route and time of delivery.
Conclusion The remarkable proliferative and differentiation capacity of stem cells promises an unlimited supply of specific cell types including viable functioning cardiac cells. ESC/iPSC-derived cardiomyocytes represent a suitable cell source that may play a major role in regenerative therapy in the future. However, ES/iPS cell technology has several key issues that remain to be overcome, including the low efficiency of cardiac myocyte differentiation from pluripotent stem cells, the low retention and survival rate of cells when injected or infused into the myocardium, and the possibility of tumor formation in vivo.
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Solving these issues in suitable large-animal preclinical models will be essential for successfully translating ESC/iPSC technology to improve the health of millions of human patients suffering from heart disease.
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