|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2009) 33, 325336 (Printed in Great Britain)
Embryonic stem (ES) cell-derived cardiomyocytes: A good candidate for cell therapy applications
Human Embryonic Stem Cell Program, Manipal Institute of Regenerative Medicine, Manipal University Branch Campus, # 10 Service Road, Domlur Layout, Bangalore 560 071, India
During the last decade, embryonic stem cells (ESC) have unleashed new avenues in the field of developmental biology and emerged as a potential tool to understand the molecular mechanisms taking place during the process of differentiation from the embryonic stage to adult phenotype. Their uniqueness lies in retaining the capacity of unlimited proliferation and to differentiate into all somatic cells. Together with promising results from rodent models, ESC has raised great hope among for human ESC-based cell replacement therapy. ESC could potentially revolutionize medicine by providing a powerful and renewable cell source capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as Parkinson's disease, myocardial infarction (MI) and diabetes. Somatic stem cells are an attractive option to explore for transplantation because they are autologous, but their differentiation potential is very limited. Currently, the major sources of somatic cells used for basic research and clinical trials come from bone marrow. But their widespread acceptability has not been gained because many of the results are confusing and inconsistent. The focus here is on human embryonic stem cells (hESCs), using methods to induce their differentiation to cardiomyocytes in vitro. Their properties in relation to primary human cardiomyocytes and their ability to integrate into host myocardium have been investigated into how they can enhance cardiac function. However, important aspects of stem cell biology and the transplantation process remain unresolved. In summary, this review updates the recent progress of ES cell research in cell therapy, discusses the problems in the practical utility of ESC, and evaluates how far this adjunctive experimental approach can be successful.
Keywords: Embryonic stem cells, Precardiac mesoderm, Characterization, Differentiation, Cardiomyocytes, Cell replacement therapy, Transplantation, Engraftment, Cardiac regeneration.
1Present address: Stempeutics Research Malaysia Sdn. Bhd., Technology Park Malaysia, Bukit Jalil, Kuala Lumpur 57000, Malaysia.
Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst-stage embryos, which are surplus to requirements for assisted reproduction (Thomson et al., 1998; Mitalipova et al., 2003). Being pluripotent in nature, these cells can be grown indefinitely in an undifferentiated state without senescence and major alteration to their genetic or epigenetic signature. They further demonstrate the capacity of differentiating into all somatic cell types of an adult body as well as extraembryonic tissues (Itskovitz-Eldor et al., 2000). These unique properties have provided added impetus to this area of research.
Among the specialized cell types that can be induced in vitro, cardiomyocytes are particularly conspicuous, giving rise to rhythmically contracting structures containing cells highly reminiscent of normal human heart cells (Kehat et al., 2001). Myocardial infarction (MI) and congestive heart failure are the leading causes of morbidity and mortality worldwide (Caplice and Gersh, 2003). Owing to the paucity of donor hearts for transplantation, and existing pharmacological approaches being insufficient to reverse progressive heart failure, establishment of alternative methods has emerged at an astonishing rate. Cell replacement therapy would consider transplantation of appropriate heart cells to the infarcted area, regenerating at least part of the dysfunctional myocardium, securing fresh heart muscle necessary to regain organ function.
Theoretically, hESC-derived cardiomyocytes are apt for the purpose of cardiac cell therapy since they demonstrate reproducible differentiation of multiple cardiac cell types in large numbers, prolonged survival and site-specific engraftment which are the keys to success in tissue replacement therapy. However, besides the ethical concerns associated with their origin being human embryos, there are a considerable number of scientific hurdles that warrant further critical investigation before they would be suitable even for clinical trials. These include evidence of efficacy by demonstrating their ability to support long-term cardiac function in animal models that have undergone cardiac injury, scaling up to cell numbers that would be adequate for treating a human heart, and issues of safety, both in terms of the potential of transplanted cells to cause arrhythmias in the host heart and for any residual undifferentiated stem cells to develop tumours.
A profound understanding of the molecular control of cardiomyocyte differentiation, which mimics the developing embryo, may facilitate addressing some of these critical problems. In this review we first consider crucial aspects of early embryonic heart development in concurrence to cardiomyocyte formation by embryonic stem (ES) cells and then review the present state of knowledge on hESC-derived cardiomyocytes. Finally, we explore how well the ES cells are going to be manipulated to serve as renewable source of functional cells to be used in cell replacement therapy for degenerative heart diseases. This review also discusses the problems encountered whilst considering ES cells as a treatment modality and also emphasizes the importance to ensue an optimal ES cell-based treatment for cardiovascular disorders.
2 Early morphogenesis in the mammalian heart
Development and acquisition of a cardiac fate are the fundamental steps in heart formation, and are the pre-requisites for further development. While commitment to cardiac lineage results from inductive interactions during early gastrulation; several discrete steps are involved in heart development in general. Truly, the well-orchestrated morphological and molecular events that result in the formation of this complex organ are intriguing. Currently, one of the biggest challenges is to identify molecules that regulate heart development and to understand their intertwined regulatory relationships. Dissecting the genetic pathways might facilitate in revealing the crucial roles of various growth factor family members in this process.
2.1 Embryonic mesoderm development
The heart is one of the first functional organs to be established in the vertebrate embryo and is important for the survival of the embryo. At the onset of gastrulation, the epiblast cells in the posterior part of the embryo undergo an epithelial to mesenchymal transition and form a transient structure known as the primitive streak (PS) from which the mesoderm emerges. The newly formed mesoderm migrates away from the primitive streak and is patterned into various subpopulations with distinct developmental fates. The cardiovascular lineages originate from mesoderm (DeRuiter et al., 1992; Lawson et al., 1991; Parameswaran and Tam, 1995). The first mesodermal cells to develop within the embryo contribute predominantly to the extraembryonic tissues, giving rise to the hematopoietic and vascular cells of the yolk sac. Cardiac mesoderm is derived from epiblast cells that move through a more distal region of the PS at a slightly later point in time (Parameswaran and Tam, 1995). Furthermore, the neural crest, a transient component of the ectoderm situated between the epidermis and the neural tube also plays an important role to heart development in concert with the mesoderm component. The cardiac neural crest overlaps the vagal neural crest and migrates to populate the pharyngeal arches 3, 4 and 6 (producing structures in the head) and to the heart, forming connective tissue that separates the great vessels of the heart.
2.2 Morphogenesis of the heart
At embryonic day (E) 7.75 of development, these mesoderm cells migrate to the anterior region of the embryo, as an epithelial layer to form a structure at the cranial border of the disc known as the “cardiac crescent” or primary heart field (DeRuiter et al., 1992). Once formed, the cardiac crescent fuses at the midline to form the heart tube consisting of an outer layer of myocardium which harbors not only progenitors of the atrial, ventricular and outflow tract lineages, but also and an inner layer of endothelial cells, a population known as endocardium. At E8.25 the heart tube through a series of “ballooning” steps and morphogenetic changes, ultimately forms the four-chambered heart (Kattman et al., 2007; Wagner and Siddiqui, 2007).
2.3 Transcription control during cardiac development
One of the earliest markers defining the mesoderm lineage is the T-box transcription factor (TF) brachyury (Kispert and Hermann, 1993; Kispert and Herrmann, 1994). Brachyury or T is expressed in all nascent mesoderm and downregulated as these cells undergo patterning and specification. Recently, Isl1, a LIM homeodomain transcription factor, has been shown to label cardiac progenitors (Moretti et al., 2006) that proliferate prior to differentiation and contribute for the majority of cells to the heart (reviewed in Laugwitz et al., 2008). One of the earliest TFs to be expressed in the crescent and crucial for heart development is the homeodomain factor tinman/Csx/Nkx2.5 (Bodmer, 1993; Komuro and Izumo, 1993; Lints et al., 1993; Lyons et al., 1995). Another important TF is the GATA transcription factor family members, GATA-4, -5, and 6, which are broadly expressed in gut epithelium, in precardiac mesoderm, and heart in regions overlapping with Nkx2.5 (Heikinheimo et al., 1994; Morrisey et al., 1996, 1997). Members of the T-box family of TFs, including Tbx1–5, 18, and 20, are also crucial for cardiac specification (Xu et al., 2004; Merscher et al., 2001; Plageman and Yutzey, 2005). Nkx2.5 and Tbx5 together with members of the GATA family of zinc finger transcription factors and with serum response factor (SRF) to activate cardiac structural genes, such as actin, myosin light chain (MLC), myosin heavy chain (MHC), atrial natriuretic factor (ANF), troponins and desmin. Members of the myocyte enhancer factor 2 (MEF-2) family of transcription factors also participate in cardiomyocyte differentiation by triggering cardiac constitutive genes. Thus multiple complex interactions take place between various transcription factors to control initial differentiation and maturation of cardiomyocytes.
2.4 Tissue interaction as an effective moderator of cardiogenic induction
The generation of functional cardiomyocytes in embryos is influenced by a combination of positive and negative induction signals produced from adjacent tissues, as demonstrated using avian and amphibian systems (Lough and Sugi, 2000; Nascone and Mercola, 1995). Anterior endoderm in particular appears to have an instructive function in cardiogenesis in various species (reviewed in Brand, 2003). Noticeably, certain endoderm-derived growth factors such as activin A, insulin, fibroblast growth factor 1 (FGF1), FGF-2, FGF4, bone morphogenetic protein (BMP) 2 and 4 possess the ability to induce proliferation of precardiac mesoderm and enhance cardiac development both in vivo and in vitro (Alsan and Schultheiss, 2002; Barron et al., 2000). The BMPs in concert with FGFs also stimulate the upregulation of cardiac transcription factors GATA-4 and Nkx2.5 expressed in the precardiac mesoderm (Schultheiss et al., 1997; Andrée et al., 1998). Further, Wingless in Drosophila and related Wnt proteins in vertebrates are involved in cardiac specification although their function is complex.
Likewise, using ES cell differentiation, as an in vitro model to derive cardiomyocytes, we have demonstrated that endoderm derivatives contribute to the differentiation of cardiomyocytes in mouse and human ES cells via BMP, FGF, Smad and Wnt signaling pathways (Pal and Khanna, 2005, 2006, 2007). The effect of these growth factors together on cardiac differentiation appears to be temporally regulated. Thus, different growth factor families interact with each other in the early steps of cardiogenesis in a time- and concentration-dependent manner (Klaus et al., 2007; Tzahor, 2007; Srivastava, 2006).
3 Differentiation of ESC to cardiogenic lineage
Implementation of the multi-faceted applications of ESC would be greatly facilitated both by derivation of a range of hESC lines that represent a cross-section of genetic diversity within the human population and by establishment of generic differentiation protocols that function between lines. However, since varying culture parameters like media components, cell plating density, number of days in differentiation, propensity of the hES line and other factors may influence the outcome, it is provoking to examine the broad applicability of specific differentiation protocols.
3.1 In vitro derivation of cardiomyocytes from mouse ESC
Pluripotent mouse embryonic stem cells (mESC) derived from mouse blastocysts (Evans and Kaufman, 1981) maintain the capacity to give rise to derivatives of the three germ layers in culture. In vitro differentiation is commonly induced by withdrawing leukemia inhibitory factor (LIF), through formation of aggregates known as embryoid bodies (EBs). EBs essentially contain a broad spectrum of cell types representing derivatives of the primary germ layers and morphologically resemble the extraembryonic yolk sac. After a few days of culture under appropriate conditions of cell density, culture medium along with growth supplements, appearance of spontaneously contracting cardiomyocytes can be observed (reviewed by Boheler et al., 2002). Differentiation may be augmented by inducing agents such as DMSO or retinoic acid or co-culture with endoderm-like cells (Mummery et al., 2002; Rathjen et al., 1999; Rathjen and Rathjen, 2001). Cardiomyocyte differentiation in mEBs recapitulates the sequential expression of cardiac genes observed in the mouse embryo in vivo. Similarly, our group has underlined the role of FGF-2 in differentiation of mESC into beating cardiomyocytes by inducing the upregulation of a candidate set of signaling molecules such as BMP-2/4/5/7, FGFR1/2 and PKC (Pal and Khanna, 2005, 2006). GATA-4 and Nkx2.5 transcripts appear before mRNAs encoding ANF, MLC-2v, α-MHC and β-MHC.
Further, the organization of the sarcomeric proteins and the electrophysiological properties of the mESC-derived cardiomyocytes have also been investigated in greater detail (reviewed by Boheler et al., 2002). With progressive days in culture, the rate of contraction decreases in concurrence to normal mouse development. Subsequently, there is a distinct development myofibrillar and sarcomeric organization coupled with abundance of functional gap junctions between cells indicating cell maturation. Terminally differentiated mESC-derived cardiomyocytes are responsive to β-adrenergic stimulation, whilst early ones are not (Maltsev et al., 1999), which is reminiscent of isolated fetal or neonatal cardiomyocytes.
However, due to the paucity of appropriate cell-surface markers reactive to cardiomyocytes or their precursors, it was imperative for initiating studies in order to transplant pure cell populations of specific cardiac lineages to the adult mouse heart. Towards this direction, several studies have been undertaken to select the mESC by genetic manipulation using α-MHC promoter (Klug et al., 1996), MLC-2v or α-cardiac alpha-actin promoter coupled with either neoR or GFP for selection (Meyer et al., 2000; Müller et al., 2000; Kolossov et al., 1998). However, to date the most significant advancement towards upscaling was made when 109 mESC-derived cardiomyocytes were produced by genetic selection in combination with a cardiac-specific promoter on EBs grown in a bioreactor (Zandstra et al., 2003).
3.2 In vitro derivation of cardiomyocytes from human ESC
The first report of cardiomyocytes from hESCs was in 2001, using H9.2, a clone of the parental hES line H9, via EB formation method (Kehat et al., 2001). Contracting cardiac foci was first observed at 27–30 days of differentiation. Likewise, spontaneous differentiation to cardiomyocytes in aggregates was also observed by others using different cell lines, e.g. H1, H7, H9, H9.1, H9.2 and H14 although the percentage of beating EBs exhibited tremendous variation ranging from 2 to 70% (Xu et al., 2002; He et al., 2003). Although, the reasons for these apparent differences in efficiency are elusive, it may be attributed to the unique genetic history of individual cell lines, propensity of a particular cell line towards a specific lineage and the independent culture conditions adopted by different labs to maintain undifferentiated hESCs and also the strategy of dissociation into EBs.
An alternative approach for the derivation of cardiomyocytes from hESCs was described by Mummery and co-workers (Mummery et al., 2002, 2003). Beating areas were observed following co-culture of hESCs (HES-2) with a mouse visceral endoderm-like cell line (END-2). This was in congruence with the obligate role of endoderm derivatives in the differentiation of cardiogenic precursor cells. This resulted in beating areas in approximately 35% of the cells after 12 days in co-culture (Mummery et al., 2003). Recently, SB203580, a specific p38 MAP kinase inhibitor was identified as a potent promoter of hESC-cardiogenesis in presence of END-2 (Graichen et al., 2007). Interestingly, the same group reported that END-2 conditioned medium supports cardiomyocyte differentiation from hESCs (Xu et al., 2008).
Although there have been several successful attempts to generate cardiomyocytes from hESC, the important challenge which still persists is the poor efficiency and insufficient numbers to treat adult human patients. In addition, it is extremely important to eliminate the risk of teratoma formation by reducing the co-existence of undifferentiated hESC while preparing batches prior to transplantation for clinical applications. Further, significant upscaling will be required to produce cardiomyocytes in sufficient numbers. This could involve increasing the efficiency of cardiomyocyte differentiation, promoting proliferation of the emerging cardiomyocytes or developing methods of purification of the required cardiac cell type. The only enrichment method described to date for hESC-derived cardiomyocytes employed discontinuous Percoll gradient purification (40.5 over 58.5%; Xu et al., 2002). Flow cytometric analysis and sorting could be the useful approaches for quantification and selection. However, lack of suitable cardiomyocyte specific cell-surface markers may restrict the widespread application of this approach.
3.3 Enrichment of cardiac differentiation from hESC
It is evident that variety of proteins governs cardiogenesis in a well-orchestrated manner and this phenomenon is highly conserved in several species. Overall, 3 families of peptide growth factors have been widely implicated in promoting cardiogenesis, namely BMPs, members of the TGF-β superfamily, the Wnts and the FGFs. Despite differences in the morphology, growth kinetics and molecular signaling pathways between mES and hESCs, studies on cardiomyocyte differentiation from mouse ES cells might help us in obtaining important insights into cardiac differentiation of human ES cells. However, several of these potential cardiogenic factors like DMSO and retinoic acid in mESCs, produce no significant improvement in cardiomyocyte differentiation (Kehat et al., 2001; Xu et al., 2002). Treatment of hESCs with 5-aza-2′-deoxycytidine, a demethylating agent showed a time- and concentration-dependent effect on cardiomyocyte differentiation and proliferation (Xu et al., 2002; Cui et al., 2007). The presence of fetal calf/bovine serum during differentiation also has an impact on differentiation efficiency since it contains a cocktail of factors (Bettiol et al., 2007). In most of the reports to date, serum has been present in the culture medium. Nevertheless, there is evidence of a 24-fold increase in the number of beating areas when hESCs co-cultured with END-2 cells were differentiated in the absence of serum instead of 20% fetal calf serum (Passier et al., 2005). Recently, we showed the inductive role of BMP-2 in absence of high serum content to maintain cardiac differentiation in two different cell lines (Pal and Khanna, 2007). Furthermore, Denning et al. (2006) comprehensively studied the effect of other conditions including technique of cell dissociation, feeder cell density, extracellular matrices, combinations of culture media and conditioned medium on the efficiency of cardiomyocyte differentiation in HUES-7 and BG01 cell lines. Most recently, a novel study has demonstrated the capacity of androgens to stimulate increased differentiation of mouse ES cells to cardiomyocytes (Goldman-Johnson et al., 2008), and is in keeping with recent observations that androgen receptor (AR)-deficient mice exhibit cardiac impairment in adulthood. Adopting a somewhat different approach, Xu et al. (2006) recently reported a novel culture method for enrichment of cardiomyocytes derived from hESC. Here, cardiomyocytes were isolated from EB outgrowths by Percoll separation and then enriched by culturing the aggregates of cells (termed cardiac bodies, CBs) in suspension. Enrichment of cardiomyocytes was evident by the increase in the expression of cardiac α/β-MHC and cTnT to 35–66% in CBs in suspension culture compared to unpurified EB outgrowths However, variations in concentration, timing and synergistic effects of potential cardiogenic factors in hESC differentiation process might have a telling effect on the outcome of cardiomyocyte differentiation.
Better strategies to overcome the high degree of heterogeneity of differentiated populations are required. Anderson et al. (2007) showed the utility of two transgenic approaches in enrichment of cardiomyocytes derived from HUES-7 cells. Negative selection of proliferating cells has been shown with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system; and secondly by positive selection of cardiomyocytes expressing a bicistronic reporter [green fluorescent protein (GFP)-internal ribosome entry site (IRES)-puromycin-N-acetyltransferase (PAC)] from the human α-myosin heavy chain promoter. Interestingly, parental and transgenic HUES-7 cells were similar with regard to morphology, pluripotency marker expression, differentiation, and cardiomyocyte electrophysiology. This, to date, is the first report on genetic manipulation of hESC towards cardiac lineage.
4 Characteristics of hESC-derived cardiomyocytes
The preceding sections have discussed the existing protocols to promote spontaneous and induced differentiation of hESCs towards a cardiac fate. Although functional cardiomyocytes can easily be distinguished in vitro by their beating phenotype, only in-depth examination can establish the specific cardiogenic cell types produced, the stages they represent pertaining to differentiation, compared to in vivo cardiac development. It is also necessary to investigate their functional properties, respecting the contractile function, underlying intracellular Ca2+ handling and whether they respond appropriately to pharmacological agents.
4.1 Molecular and cellular characterization of cardiomyocytes
Upon commitment towards the cardiac lineage, a unique molecular signature comprising of early stage transcription factors, structural/granular proteins and metabolic regulators facilitates distinction between undifferentiated hESC and their differentiation derivatives. This finding is well correlated to the early stages of heart development. Gene and protein expression analysis of hESC-derived cardiac myocytes has demonstrated the presence of cardiac transcription factors including Brachyury, HAND1, GATA-4, MEF-2, and Nkx2.5 (Kehat et al., 2001; Pal and Khanna, 2007). Simultaneously, structural elements of the myofibres such as α-, β- and sarcomeric-MHC, atrial and ventricular forms of MLC, tropomyosin, cardiac troponin I and T, α-actinin and titin are also highly expressed. Additionally, upregulation of ANP, a hormone that is constitutively expressed in both atrial and ventricular cardiomyocytes in the nascent heart, has also been observed during cardiac differentiation of hESCs (Pal and Khanna, 2007). However, a significant development has been the global transcriptome analysis of mouse and human ESC-derived cardiomyocytes (Doss et al., 2004; Filipczyk et al., 2007).
Although, in general hESC-derived cardiomyocytes fail to express non-cardiac markers, they react with antibodies to smooth muscle actin (SMA) and myoglobin (Xu et al., 2002). This is not surprising since both of these markers are associated with the formation of the mesoderm lineage. Immunofluorescence studies on differentiated cardiomyocytes reveal sarcomeric striations organized in separated bundles, which closely mirror the innate pattern seen in human fetal cardiomyocytes and not the well-organized parallel bundles commonly found in human adult cardiomyocytes (Mummery et al., 2003). Furthermore, maturation takes place at least in part in hESC-derived cardiomyocytes, seen in ultrastructural examination as organized myofibrillar bundles, intercalated discs, sarcomere, gap junctions and desmosomes (Kehat et al., 2001). This is supported by the identification of distinct sarcomeric organization in cultures maintained for 50 days or more (Snir et al., 2003). Also the terminally differentiated cardiomyocytes proliferate remarkably slowly compared to their younger counterparts, which may be helpful in bypassing the potential risk of teratoma formation post-implantation in vivo.
4.2 Functional characterization of cardiomyocytes
Although cardiomyocytes can be efficiently generated from hESCs ex vivo, whether a functional syncytium can be formed between donor and recipient cells after engraftment has evoked considerable debate. It is necessary that the transplanted cells fully integrate within the diseased myocardium contribute to its contractile performance, and respond appropriately to various physiological stimuli (e.g. β-adrenergic stimulation). Using a combination of electrophysiological, imaging, and molecular techniques, certain groups of researchers have carried out extensive work in an attempt to establish this hypothesis.
The hESC-derived cardiomyocytes innately express adhesion molecules (N-cadherin) and the gap junction proteins connexin-43 and 45 substantiating the formation of electromechanical coupling ex vivo (Mummery et al., 2003). Perhaps, the best-studied receptors that increase cAMP are the β-adrenergic receptors. Stimulation of β-adrenergic receptors via endogenous catecholamines (e.g., norepinephrine, epinephrine) or synthetic agonists (e.g., isoprenaline, isoproterenol) promotes adenylyl cyclase activation, leading to an increase in intracellular cAMP. This in turn is followed by an activation of cAMP-dependent protein kinase A (PKA), which phosphorylates specific substrates, including some TFs and proteins closely associated with cardiac function such as brachyury, GATA-4, HAND1 and cripto1. Pharmacological induction with phenylephrine (α1-AR agonist) and isoprenaline (β1-AR agonist) showed a dose-dependent enhancement of the contraction rate in both human fetal and hESC-derived cardiomyocytes (Kehat et al., 2001; Mummery et al., 2003; Denning et al., 2006; Pal and Khanna, 2007). Inhibition of phosphodiesterase by isobutyl methyl xanthine (IBMX) and activation of adenylate cyclase by forskolin results in increased or decreased beat rate, respectively (Kehat et al., 2001). In contrast, acetylcholine, exerting muscarinic effect in the myocardium, reduced the contractile activity in the hESC-derived cardiomyocytes in a concentration-dependent manner (Norstrom et al., 2006). However, variable responses may be exhibited by the cardiomyocytes maintained at different stages in differentiation mimicking mouse embryonic development (Wobus et al., 1991).
When cultured in a suitable tissue culture environment with an appropriate Ca2+ concentration and electrolyte balance, the cardiomyocytes differentiated from hESC show spontaneous periodic contractile activity. In fact, the main currents engaged in the action potential in heart muscle are influx of Ca2+ and Na+ during depolarization and efflux of K+ during repolarization and resting potential. Sodium channels are the major current of depolarization in atrial and ventricular cells but in pacemaker cells it is Ca2+ via L-type calcium channels (Mummery et al., 2003). L-type calcium channels are the predominant route for calcium entry into cardiac myocytes, and are key components in excitation–contraction coupling. Compounds that reduce available Ca2+ concentration or otherwise interfere with transmembrane transport of Ca2+ often affect contractile activity. Likewise, in hESC-derived cardiomyocytes the L-type calcium channel blocker diltiazem inhibits contraction in a dose-dependent manner (Xu et al., 2002). Verapamil, a blocker of the transmembranous flow of Ca2+ ions and an inhibitor of mobilization of intracellular Ca2+ also reduced or abolished the contractile activity (Norstrom et al., 2006).
In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial (SA) node. Ca2+ release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. Similarly, a depressed rate of pacemaker potential was produced in ES-derived cardiomyocytes by ryanodine, and rescued in knockout (KO) myocytes, devoid of a full-length RyR2 (Yang et al., 2002). In contrast, Binah et al. (2007) reported that RyR and thapsigargin do not affect the Ca2+ transient and contraction, suggesting that at this developmental stage, the contraction does not depend on sarcoplasmic reticulum Ca2+ release. It was also demonstrated by this group that although hESC-derived cardiomyocytes express SERCA2 and Na+/Ca2+ exchanger (NCX) at levels comparable to those of the adult human myocardium, calsequestrin and phospholamban are not expressed. This suggests that the mechanical function related to intracellular Ca2+ handling of in vitro cardiomyocytes differs from the adult myocardium, probably because of immature sarcoplasmic reticulum capacity. On the other hand, repolarization is initiated by numerous K+ channels. Among them, transient outward current – which causes the early rapid repolarization – is encoded by the genes Kv4.2, Kv4.3 and KvLQT1. Transient expression of these cardiac ion channel genes has been detected in hESC-derived cardiomyocytes in a time-dependent manner (Mummery et al., 2003).
Last, electrophysiology can be studied by patch-clamp analysis for cardiomyocyte action potentials, such as resting potential, upstroke, amplitude and duration. Patch-clamp electrophysiology on dissociated hESC cardiomyocytes showed that different electrical phenotypes were present (Mummery et al., 2003; He et al., 2003). Ventricular-like action potentials predominated, but atrial-like, pacemaker-like and vascular smooth muscle-like cells were also found. In spite of certain anomalies, these findings were comparable to early stages of fetal development.
5 Cell replacement therapy
Degenerative diseases such as myocardial infarction (MI), Parkinson's disease, and diabetes are due to the progressive loss of functional resulting from cell injury or degeneration. Replenishing the damaged cells by fresh functional cells in order to restore the normal function of the tissues or organs is the underlying principle of cell replacement therapy or alternatively regenerative medicine. It is expected that the organs or tissues treated by this approach can salvage their normal function more efficiently than the ones treated by conventional therapies like organ transplantation and pharmocological treatment or the use of ventricular assisted devices. The practical utility of this approach primarily depends upon the availability of a versatile source of judicially selected cells.
5.1 Suitability of ESC in cardiovascular therapy
Cardiovascular disease is the major cause of mortality and morbidity in the developed as well as in developing countries. In the Indian subcontinent, cardiovascular disease is responsible for more than 25% of deaths. It is further predicted that in the next 15 years this country alone will host more than half the cases of heart disease in the world (Gupta et al., 2008). Determining the etiology of heart failure can be complex due to a lack of consistent diagnostic criteria and patients frequently complaining of multiple morbidities. Owing to the limited capacity of residing cardiomyocytes, the myocardium in particular is vulnerable to irreversible injury and poor outcome (Towbin and Bowles, 2000). The injured myocardium is replaced by a fibrous scar impairing ventricular function. Recently, however, stem cell-based therapy has become a realistic option to replace damaged cardiomyocytes. A number of different cell types are currently being evaluated which include, cell therapies derived from primary cell isolates, established cell lines, adult stem cells namely bone marrow and mesenchymal stem cells, cord blood stem cells and of course ESC-derivatives (reviewed in Fodor, 2003). Several strategies of cell transplantation such as intramyocardial, intracoronary, transendocardial/transepicardial and intravenous injections (Collins et al., 2007) are being optimized. Nevertheless, ESC emerges as the most promising candidate in cell replacement therapy for heart disorders simply because they demonstrate reproducible differentiation, prolonged survival and site-specific engraftment. Furthermore, the upcoming experimental results with human ES cells have proven to be encouraging and promising (Kehat et al., 2004; Laflamme et al., 2005; Xue et al., 2005; Kofidis et al., 2006). These results have added momentum to this approach (Fig. 1).
Embryonic stem cell therapy for cardiac disorders.
5.2 Pre-clinical studies with ESC-derived cardiomyocytes
The basic question being addressed in pre-clinical experiments is whether ex vivo or in vivo expansion might be the most suitable approaches in increasing their numbers and improving contractile function of the heart. Subsequently, ESCs are developmentally the most versatile of stem cells forming all of several hundreds of cell types in the adult body. Among the most important issues being considered is to identify the most suitable stem cells for replacing cardiac muscle and to determine which mechanisms contribute to stem cell-mediated improvement in cardiac function after MI.
5.2.1 Cell transplantation
Although, ESCs appear to be extremely promising as a potential new therapeutic strategy, several obstacles need to be overcome before clinical application of hESC-derived cardiomyocytes becomes a reality. The most efficient way to differentiate pluripotent cells into a homogenous population and the site to deliver them still needs to be determined. The likelihood of teratoma formation and graft rejection are 2 very important issues in ESC transplantation. Furthermore, the fate of transplanted ESC-derivatives would have to be examined for longer time-points in terms of toxicity and efficacy. Importantly, several groups report successful transplantation of mESCs in terms of cell survival, site-specific engraftment, proliferation, further differentiation and improvement of cardiac function by contractile activity, LVEF, LVDF, etc. (Klug et al., 1996; Yang et al., 2002; Himes et al., 2004; Hodgson et al., 2004; Kofidis et al., 2004; Min et al., 2002; Naito et al., 2004; Singla et al., 2006). However, experience with hESC transplantation remains limited.
5.2.2 Animal models
The majority of in vivo experiments have focused on the capacity of stem cells to restore regional ischemic injury in the heart post-ligation of the coronary artery or inducing cryoinjury. Small animals or rodents have mainly been used for transplantation of ESCs into either uninjured (Klug et al., 1996; Reinecke et al., 1999; Naito et al., 2004; Laflamme et al., 2005; Kofidis et al., 2004) or infarcted (Etzion et al., 2001; Yang et al., 2002; Hodgson et al., 2004; Himes et al., 2004; Kofidis et al., 2006; Leor et al., 2007; Singla et al., 2007; Dai et al., 2007; Laflamme et al., 2007) hearts. Nevertheless, larger animals, including primates, are going to be necessary for testing of efficacy of ES-derived cardiomyocyte therapies before clinical trial commence in human. Studies in mice or rats may not be reliable in predicting transplanted cells generating arrhythmias because of their exceptionally high heart rate. Notwithstanding, a few groups have been successful in achieving thrombolysis in myocardial infarction (TIMI) in sheep (Ménard et al., 2005) and an AV-block in a swine (Kehat et al., 2004) to evaluate the role of ESC-derived cardiomyocytes. However, chronic heart failure can be caused not only by ischemic injury but also by diffuse cardiomyopathy. By using a doxorubicin (DOX)- induced model for cardiomyopathy in rodents (Schwarz et al., 1998) the potential role of stem cells has established by demonstrating cardiomyocyte integration in vivo and alleviation of physiological activity of the damaged heart (Agbulut et al., 2003; Ishida et al., 2003). Notably, a familiar side effect of prolonged DOX-therapy is the development of congestive heart failure, which is attributed primarily to cardiomyocyte apoptosis due to increased oxidative stress and depleting endogenous antioxidants (Kalyanaraman et al., 2002).
5.2.3 Methods of cell delivery
Intramyocardial injection of cells for cardiomyoplasty involves direct injection of 1–10 million ESCs into the myocardium of the infarcted region with a small needle depending on the size of the animal. This delivery system has been commonly used in a wide range of studies on cell therapy (Etzion et al., 2001; Hodgson et al., 2004; Kofidis et al., 2006; Laflamme et al., 2007). Although intramyocardial injection enables cell implantation at the damaged site of an affected heart, there are certain drawbacks, including low percentage of cells delivered, possible arrhythmogenicity, and the need for an open-heart surgery. An alternative approach may be cardiomyocytes grown on a biodegradable matrix or membrane or film such as fibrin, poly-lactic acid (PLA), poly-lactic-glycolic acid (PLGA) and implanted as a “cardiac patch” at the site of injury.
Intracoronary transplantation is a catheter-based technique and has gained considerable attention since cells are delivered directly to the infracted myocardium without having to travel through the systemic circulation. However, questions have been raised about the poor rate of engraftment (1–2%) following this method. In addition, transepicardial injection of bone marrow-derived mononuclear cells enhances angiogenesis and reduces infarct size in pigs (Waksman et al., 2004), whereas transendocardial injection is performed under the guidance of electromechanical mapping. Furthermore, it has been recently reported that intravenously infused donor cells can migrate and engraft to the site of injury in animal models of MI (Min et al., 2006) and murine myocarditis (Malek et al., 2006).
5.2.4 Tumor formation
Implantation of undifferentiated ESC leads to formation of benign teratoma in the recipients (Thomson et al., 1998). This demands a pure population of terminally differentiated cell phenotype. New strategies and methodologies need to be developed to isolate and propagate the terminally differentiated cells which are likely to be post-mitotic. However, transplantation of undifferentiated hESC has been reported to survive in the rat hearts during MI without the formation of teratoma (Xie et al., 2007). The mechanism involved may be attributed to the paracrine effect of the partially damaged myocardium. Likewise, a similar mechanism may become activated during cardiomyocyte transplantation. For example, growth factors such as TGF-β can promote in vivo organ-specific differentiation of early ESCs, via stimulation of paracrine pathways eventually leading to improvement of myocardial function after cell transfer into an area of ischemic lesion (Kofidis et al., 2005).
5.2.5 Immunogenic reaction
Before safe clinical applications, the immune properties of ESC-derived cardiomyocytes need to be properly understood. It has been postulated that hESCs, like mESCs (Tian et al., 1997), lack MHC protein expression and, therefore do not trigger an immune response in the host. However, Drukker et al. (2002) showed that, although at low levels, hESCs do express MHC class I molecules, this expression increases upon differentiation in vitro. They later reported weak allorejection processes against ESC-derivatives (Drukker et al., 2006). Further, Li et al. (2004) demonstrated the immune privilege of differentiated ESCs, although it was subsequently found that their immunogenicity of mESC increases upon differentiation after transplantation into ischemic myocardium (Swijnenburg et al., 2005). Conversely, the myocardium may be a relatively hospitable environment in terms of immune response (Drukker and Benvenisty, 2004).
Different experimental designs may explain the disparate results. First, different ESC lines (mESCs vs. hESCs) were used; second, the cells were injected in different target tissues (normal vs. infracted tissue). However, the immune properties of ESC-derivatives are likely to be altered in MI microenvironment in presence of an aggravated inflammatory cell infiltration. As a result, immunosuppressive therapy appears to be an option when ESCs or their derivatives are used in treatment of MI. Further, immunodeficient mice and their immunocompetent counterpart display different degree of immune reaction post-transplantation (Yang et al., 2002; Kofidis et al., 2005). In a study with mESCs, cyclosporin was administered to rats in order to prevent immune rejection (Naito et al., 2004). Furthermore, hESCs were less susceptible to immune rejection than adult cells, even when differentiated (Laflamme et al., 2005; Kofidis et al., 2006). Besides the microenvironment surrounding ESC grafts, some features of the ESC-differentiated cardiomyocytes might also contribute to an increased immunogenicity. ESC-derived cardiomyocytes are in fact a kind of fetal cardiomyocyte-like cells and thus might acquire higher immunogenicity in later developmental stages (Goh et al., 2005). Further, the co-existing undifferentiated ES cells in cardiomyocytes might induce immune responses, as no strategy till date has been shown to produce purely homogeneous differentiated population. In addition, these contaminating ESCs in vivo are likely to develop into multiple cell lineages (Singla et al., 2006) capable of inducing severe immune responses.
Because disparities exist among hypotheses concerning the immunogenicity of ESCs and their derivatives, several strategies in prevention of potential immune rejections against transplanted stem cells are under rigorous experimentation. Microchimerism may be a possible option to induce donor specific tolerance; this could be achieved in several ways among which intraportal ESC injection has been recently used (Fändrich et al., 2002) wherein rat ESC-like cells (RESC) were grafted into fully MHC-mismatched rats, which survived without immune rejection for over 150 days. The authors further argued that T-cell ignorance and deletion involving FasL expression might be the underlying mechanisms for the RESC-induced tolerance (Fändrich et al., 2002). An improved way to reduce or eliminate graft rejection is banking of hESC lines with a range of HLA profiles or induction of immunotolerance in the recipient (Drukker and Benvenisty, 2004; Civin and Rao, 2006). Further, isogenic ESCs or patient-specific stem cell lines can be created by the technique of transferring somatic nucleus to an enucleated oocyte (Tada et al., 2001). To create a universal donor cell line is another appealing option to meet the basic goals of stem cell banks. Methods to reduce immune rejections, such as β2-microglobulin gene knockout (Zijlstra et al., 1990) and FasL expression induction (Griffith et al., 1995), have also been proposed. Nonetheless, the unsuitability of such techniques in terms of tedious methodology, lack of reproducibility and consistent results calls for further investigation before ESCs can be exploited in repair medicine.
5.2.6 Myocardial tissue regeneration and restoration of cardiac function
ES cells evidently represent a potentially valuable and renewable source of cells that can be used for transplantation therapy. However, the specific mechanism(s) by which cell-based therapy causes beneficial effects on cardiac function remain elusive. Nonetheless, several studies have demonstrated enhanced cardiac function after MI, sometimes even sustained, in animals following ES cell transplantation but the transplanted cells were barely detectable implicating poor survival and engraftment in situ. However, in some cases heart remodeling and extracellular matrix deposition appeared altered, so unknown paracrine mediators have been proposed as key players in functional improvement (Dowell et al., 2003; Laflamme and Murry, 2005; Dai et al., 2005; Singla et al., 2007). In a recently published paper, Laflamme and colleagues have identified a cocktail of pro-survival factors consisting of growth factor-reduced Matrigel, supplemented with ZVAD (benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethyl ketone), Bcl-XL, cyclosporine A, IGF-1 and pinacidil, that limits cardiomyocyte death after transplantation (Laflamme et al., 2007). They generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation in presence of activin A and BMP4. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium not only attenuated ventricular dilation but also preserved regional and global contractile function after MI compared with controls receiving non-cardiac hESC-derivatives or vehicle. Furthermore, augmentation of blood vessel formation has frequently been observed concomitant with rescue of the myocardial tissue in the infarcted region (Kocher et al., 2001; Laflamme et al., 2007).
The primary objective of cardiomyocyte transplantation is to improve cardiac performance following heart failure. Methods of assessing heart function include ECG (Hodgson et al., 2004), measurements of cardiac pressure (Yang et al., 2002), echocardiography and electrophysiological mapping (Kehat et al., 2004; Xue et al., 2005; Cai et al., 2007; Laflamme et al., 2007). Magnetic resonance imaging (MRI) has also been carried out to detect transplanted mESCs (Himes et al., 2004) and hESCs (van Laake et al., 2007). Each of these methods has its advantages and disadvantages. While ECG is inexpensive and widely available, it fails to assess dynamic functions of the heart. In contrast, direct pressure measurements provide more detailed information on left ventricular function, but are limited unless combined with volume measurements, which makes it technically more challenging. Echocardiography and MRI are both effective techniques since they present direct and easily interpretable images of both cardiac kinetics and morphology.
However, it is unlikely that the improvement of cardiac output in the animal models can solely be attributed to the limited number of differentiated cardiomyocytes observed in vivo. It is indeed a complex spectrum of effects of the ESC-derivatives that lead to the physiologic improvement and reduction in myocardial impairment. The donor cells stimulate a significant enhancement of endogenous repair by recruitment of resident stem/progenitor cells or as a result of some other unknown mechanism. Furthermore, the cardiac grafts perhaps induced a brisk, host-derived angiogenic response, which might have contributed partly, producing a likely increase in cardiac muscle healing.
6 Concluding remarks
This review illustrates the complicated mechanisms underlying recovery from MI and the various ways in which transplanted cells might give benefit. It is evident that utilization and practical application of ESC in cell replacement therapy are still in an “embryonic stage” and need extensive investigation and clinical trials before they can be accepted as an ideal substitute for the treatment of degenerative diseases. Nevertheless, the daily increase in experimental findings is reinforcing the hope that ESC will indeed emerge as a versatile source of renewable cells for application in cell replacement therapy. It is clear that reproducible differentiation, effective transplantation, prolonged survival and site-specific engraftment are the keys to success in cell therapy. Therefore, there is enough optimism among the scientists that ESC-based therapies may offer reliable and cost-effective therapeutic substitute for treatment of severe degenerative disorders in the near future.
7 Forward path
Cell therapy in the treatment of MI is a relatively new and exciting concept that has been studied using numerous cell types, animal models, delivery systems, and strategies. Although considerable progress has been made since the concept was first introduced, there are numerous practical hurdles that are yet to be addressed before this technology can be effectively instituted in a large patient population. The following represents a brief list of critical issues that need special attention in case of ESC-based approaches:
● Ethical concerns, logistics and economics ● Uncertain regulatory requirements
Ethical concerns, logistics and economics
Uncertain regulatory requirements
● To obviate the use of xeno-components in hES cell cultures ● Genetic and epigenetic stability of hES lines over longer time periods ● Following GMP norms
To obviate the use of xeno-components in hES cell cultures
Genetic and epigenetic stability of hES lines over longer time periods
Following GMP norms
● Uncontrolled cell proliferation ● Reprogramming ESCs viz. SCNT ● Tackling immune rejection/need for immunosuppressive regimens
Uncontrolled cell proliferation
Reprogramming ESCs viz. SCNT
Tackling immune rejection/need for immunosuppressive regimens
● Optimization of a generic differentiation protocol and its empirical testing ● Better understanding of the molecular processes governing ESC differentiation ● Large-scale production of desired cell type with appropriate functionality
Optimization of a generic differentiation protocol and its empirical testing
Better understanding of the molecular processes governing ESC differentiation
Large-scale production of desired cell type with appropriate functionality
● Optimal number of cells for transplant ● Modification of less invasive delivery systems ● Techniques to label cells for transplant and subsequent tracking of cell fate ● Long-term cell survival and site-specific engraftment ● Homing of transplanted cells in diffused cardiomyopathy ● Multiple mechanisms of action in improvement of cardiac function ● Angiogenesis and vascularization of engrafted tissue
Optimal number of cells for transplant
Modification of less invasive delivery systems
Techniques to label cells for transplant and subsequent tracking of cell fate
Long-term cell survival and site-specific engraftment
Homing of transplanted cells in diffused cardiomyopathy
Multiple mechanisms of action in improvement of cardiac function
Angiogenesis and vascularization of engrafted tissue
The author gratefully acknowledges the support in form of encouragement from all the members of Embryonic stem cell group at Manipal Institute of Regenerative Medicine, Manipal University and Stempeutics Research Private Limited, Bangalore. The author would also like to thank Dr. Aparna Khanna, Avesthagen, Bangalore for contributing through constructive criticism and fruitful discussions during the preparation of the article.
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Received 2 August 2008/24 October 2008; accepted 5 December 2008doi:10.1016/j.cellbi.2008.12.001