The introduction of human embryonic stem cell (hESC) lines provided scientists with the means for investigation of early human development and potential tools for tissue replacement thera- pies.1 Although research using hESCs suffers from significant restraints, important accom- plishments have already been achieved. This review will focus on the derivation of cardiomy- ocytes from hESCs and their potential applica- tions for the regeneration of functional myocardium.
All stem cells, whether from embryonic or adult sources, share a number of characteristics.
First, they are capable of self-renewal, meaning they can generate stem cells with similar proper- ties. Second, the stem cells are clonogenic, meaning that each cell can form a colony in which all the cells are derived from this single cell and have identical genetic constitution.
Third, they are capable of differentiation into one or more mature cell types.
These properties of stem cells make them a unique and a powerful tool both for clinicians and basic science researchers. Except for the attractive and promising potential use of stem cells for tissue regeneration, stem cells also pro- vide scientists with a unique insight to develop- mental stages that were previously out of reach for experimentation, especially in human mod- els. Moreover, better understanding of the mechanisms involved in early stem cell differen- tiation may be a prerequisite for safe and effi- cient application of stem cells in regenerative medicine.
Derivation and Differentiation of Embryonic Stem Cells
Embryonic stem cells were initially isolated from mouse blastocysts in 1981 by two inde- pendent groups.2,3 The potential therapeutic applications and the unique opportunity to study early mammalian development exempli- fied by the murine model motivated researchers to establish similar hESC lines.1,4The origin of the hESC lines, similar to the mouse and rhesus models, is from the preimplantation embryo produced by in vitro fertilization for clinical purposes and donated by individuals after informed consent. The blastocyst contains an outer cell layer and an inner cell mass (ICM).
Whereas the outer cells become the trophecto- derm and subsequently give rise to the placenta and other embryonic supporting tissues, the ICM cells will ultimately create all tissues in the body [Figure 3.1 (see color section)]. The hESC lines were established by isolating the ICM cells after removal of the trophectoderm with specific antibodies (immunosurgery). The cells were then plated on a feeder layer of mitotically inactivated mouse embryonic fibroblasts (MEFs). Plating of the hESCs on top of this feeder layer allows con- tinuous undifferentiated propagation of these cells in cultures, a unique property not shared by adult stem cells. The undifferentiated hESCs were also shown to express specific surface markers, such as stage specific embryonic antigen 3 (SSEA-3), SSEA-4, Tra-1-60 and Tra-1-81, the
3
Regeneration of the Functional Myocardium Using Human Embryonic Stem Cells
Oren Caspi and Lior Gepstien
33
embryonic transcription factor Oct-4, and alka- line phosphatase. In addition, undifferentiated hESCs express high levels of telomerase activity and retain a normal karyotype for several passages.1,4
There are many differences between hESCs and their murine counterparts, as extensively reviewed by Pera and Trounson.5Briefly, mouse embryonic stem cell (mESC) colonies are more rounded with a glassy appearance and indistinct cell borders, whereas hESC colonies are flatter and often display more distinct cell borders. In addition , the hESC doubling time is 3 times longer and it lacks the surface antigen SSEA-1 but expresses SSEA-3 and SSEA-4.6 However, the most important difference between the two cell lines is probably the mechanisms involved in their self-renewal as reviewed by Rao.7The maintenance of the undifferentiated state of the ESC is mediated both by differentiation inhibit- ing signals as well as by the lack of expression of differentiation genes. Among the differentiation inhibiting signals, the well-known leukemia inhibitory factor (LIF) is considered to be a suf- ficient trigger for the maintenance of pluripo- tency in the mESC system in the presence of serum. In addition, a recent publication suggests that induction of the expression of inhibitor of differentiation (Id) genes by bone morpho- genetic proteins (BMPs) in concert with LIF can maintain in vitro self-renewal capabilities of mESCs in serum-free conditions.8However, in the hESC system, LIF is insufficient or even not required for this purpose, and the presence of the MEF feeder layer itself, its conditioned medium9or other cell support system such as human fetal fibroblast, adult epithelial cells,10or foreskin cells,11is required.
Except for the difference in LIF-gp130 sig- naling between human and mESCs, several key regulators of stem cell self-renewal have been demonstrated to be conserved between the human and mouse systems. Sato et al.12eluci- dated the role of the canonical Wnt pathway in the maintenance of embryonic stem cell self- renewal; this study demonstrated that 6-bro- moindirubin-3′-oxime, a specific glycogen synthase kinase 3 inhibitor, is sufficient for the maintenance of “stemness” propagation and pluripotency of both human and mESCs. The homebox-domain containing protein Nanog was recently demonstrated to act parallel to the LIF pathway in maintaining ESC self-renewal both in the mouse and hESC.13–15Further stud- ies elucidating the factors participating in ESC
self-renewal are crucial for establishing a reproducible, well-defined, animal and serum- free supporting system that may be upscaled and will facilitate research practices and pro- vide a safer alternative for future clinical appli- cations of hESCs.
The most exciting properties of the hESCs lies in their potential to differentiate in vitro to cell derivatives of all three embryonic germ layers.
This ability to differentiate to a variety of mature somatic cell types was demonstrated using both spontaneous and directed in vitro differentiation systems. Since the initial report of the derivation of the hESCs, they were shown to differentiate into cardiac tissue,16 neuronal tissue including dopaminergic cells,17,18β islet pancreatic cells,19 hematopoietic progenitors,20 keratinocytes,21 bone tissue,22and endothelial cells.23
From hESCs to Cardiomyocytes
The generation of a reproducible spontaneous cardiomyocyte differentiating system from the clonal hESC line (H9.2) was originally described by Kehat et al.16in our laboratory. The induction of in vitro differentiation of hESCs to cardiomy- ocytes required their removal from the feeder layer and cultivation in suspension. The hESCs then tend to generate three-dimensional cell aggregates termed embryonic bodies (EBs). The human EBs contain tissue derivatives of endo- dermal, ectodermal, and mesodermal origin but seem to be less organized from their murine counterparts.24,25After 7–10 days in suspension, the EBs are plated on top of gelatin-coated cul- ture dishes and observed microscopically for the appearance of spontaneous contraction.
Rhythmically contracting areas appeared at 4–22 days after plating in 8.1% of the EBs.16 Recently, other groups reinforced our findings and generated hESC-derived cardiomyocytes (hESDCMs) using different clonal and non- clonal cell lines.26–28
Structural Properties
Several lines of evidence confirmed the car- diomyocyte phenotype of the contracting areas generated within the EBs. Reverse transcriptase- polymerase chain reaction studies demonstrated that cells isolated from the beating areas express the cardiac transcription factors GATA4 and
Nkx2.5 and cardiac-specific genes, such as car- diac troponin I and T, atrial natriuretic peptide, and atrial and ventricular myosin light chains (MLCs). Immunostaining studies demonstrated the presence of the cardiac-specific sarcomeric proteins such as myosin heavy chain, α-actinin, desmin, cardiac troponin I, and atrial natriuretic peptide. Electron microscopy studies revealed the development of well-aligned sarcomeres accom- panied by the development of the known sarcom- eric striated pattern with recognizable A, I, and Z bands [Figure 3.2A (see color section)].16,29 Compared with the murine ESCs,30,31 the hESCs differentiated in a slower rate and their ultra- structural development was more heterogeneous, lasted longer, and did not reach the same level of maturity.
The degree of structural and functional matu- ration of the generated cardiomyocytes may be important for the possible utilization in myocar- dial repair strategies. Although it is possible that the in vivo environment after cell grafting may promote such maturation, an alternative approach may be the application of oscillating mechanical load either in vitro or in vivo.
Zimmermann et al.32demonstrated that applica- tion of phasic mechanical stretch on neonatal rat ventricular cardiomyocytes grown as tissue constructs with collagen and other extracellular matrix factors resulted in significant maturation of the cells. In addition to mechanical stretch- ing, low-frequency magnetic fields have also been demonstrated to affect cardiac differentia- tion and might also serve to hasten the matura- tion process.33Alternative approaches might be to use certain growth factors or transcription factors to affect cell maturation such as BMP- 10,34Foxp1,35TBX5.36
The Electrophysiological Characteristics of hESDCMs
The hESDCMs were demonstrated to have a functional phenotype of early-stage human car- diac tissue including typical extracellular electri- cal activity, intracellular action potentials and ionic currents [Figure 3.2B (see color section)], calcium transients, and chronotropic response to adrenergic agents [Figure 3.2C (see color section)].
High-resolution activation maps using the multi- electrode array system demonstrated the presence of functional electrical syncytium with synchro- nous action potential propagation and pace-
maker activity (Figure 3.2D).37Immunostaining studies showed that this functional syncytium results from the presence of gap junctions [com- prised both of connexin (Cx)43 and Cx45]
between the cells.
According to studies based on the murine dif- ferentiation system, the beating EB is populated by cells representing all cardiac phenotypes. The percentage of the different cell types transforms during development from pacemaker-like cell predominance to atrial or ventricular cell pre- dominance in older EBs.31Detailed electrophysio- logical analysis of the developing murine EBs has revealed a developmental cascade of ion chan- nel expression and modulation. The noncontract- ing precursor cells display voltage-dependent L-type Ca+2 channels at very low densities.
Cardiomyocytes of an early differentiation stage exhibit a primitive pacemaker action potential generated only by voltage-dependent L-type Ca+2 channels and transient outward K+ channels.
Terminally differentiated cardiomyocytes express various additional ion channels according to their various phenotypes. Ventricle-like cells express voltage-dependent Na+ channels, delayed out- ward-rectifying K+ channels, and inward-rectify- ing K+channels. Additional ion channels, such as muscarinic acetylcholine-activated K+ channels and the hyperpolarization-activated pacemaker channels were demonstrated in atrial-like cells and sinus node-like cells, respectively.31
Similar to the mESC model, whole-cell patch clamp studies demonstrated that the hESDCMs also display cardiac-specific action potential mor- phologies and ion currents. Additional work from our laboratory revealed the basis for the sponta- neous automaticity in these cells, at least at the mid-differentiation stages. These studies revealed that, during this stage, the spontaneous electrical activity is mediated by the absence of significant inward-rectifier K+current and a prominent Na+ current sensitive to Tetrodotoxin (TTX) coupled with the presence of the hyperpolarization and cyclic nucleotide-activated ion channels (HCN) pacemaker current (If).38 The paucity of the inward current creates a high-input resistance state that allows a small inward current to bring the membrane potential to threshold. Further electrophysiological and molecular characteriza- tion of the developmental cascade of cardiac ion channels using the hESC model will provide a unique insight to the development of excitability in early human cardiac tissue and will allow engi- neering of the functional properties of the cell grafts.
Cell Proliferation
Based on animal models, cardiomyocytes switch from hyperplastic to hypertrophic growth soon after birth.39In human fetal tissue, the number of cells in the S-phase of cell cycle is estimated to be 16.8%.40 Interestingly, during the process of ultrastructural maturation of the hESDCMs, the cells start to withdraw from the cell cycle. Using [3H] thymidine incorporation or Ki-67 immunolabeling, the hESDCMs demonstrated a gradual withdrawal from cell cycle with cessation of DNA synthesis after 7 weeks.41 Several possible reasons may explain the “early” withdrawal of the hESDCMs from the cell cycle. First, disparity may stem from the inherent limitations of an in vitro model, which may be different from the in vivo setting (hemodynamic load, paracrine effect, etc.) and this may alter the cell cycle activity as well as the cell maturation. Second, the fact that the evalu- ated cells are concentrated in a small area adja- cent to nonmyocytic proliferating tissue may also activate contact inhibition signals. Because cell replacement therapy for heart failure is one of the most promising applications of the hESDCMs, the proliferative capacity of the cells is of great importance for planning the amount of cells needed for transplantation. Further studies aimed at evaluating the proliferative capacity of the hESDCMs in vivo after trans- plantation to the adult heart may provide addi- tional data concerning this issue.
Promoting Stem Cell Differentiation to Cardiomyocytes
The hESC model may provide valuable informa- tion regarding the process involved in early lineage commitment, differentiation, and matu- ration of human cardiac tissue. The currently available cardiomyocyte differentiation system of the hESC is essentially spontaneous, however, and thus characterized by relatively low efficacy and reproducibility. Understanding the mecha- nisms that drive early-cardiac differentiation of the hESC may also have important clinical implications. One of the major obstacles for the utilization of these cells in future myocardial regeneration strategies is the insufficient num- ber of cardiomyocytes achieved by the currently available differentiation scheme. Therefore, fur- ther efforts aimed at developing a more produc-
tive cardiomyocyte differentiation system are essential for the generation of the large quanti- ties of cells needed to achieve the successful application of this strategy.
The development of a directed differentiation system is hampered by the relative lack of data regarding the inductive cues that lead to com- mitment and terminal differentiation of human cardiomyocytes. Thus, strategies for directed differentiation should undoubtedly follow the research conducted in a number of model organisms, most notably the chick, amphibians, zebrafish, and the mouse. The heart arises from cells in the anterior lateral plate mesoderm of the early embryo. The cells of the cardiogenic mesoderm adopt a crescent-like morphology and are therefore termed “the cardiac cres- cent.”42The endoderm that is in direct contact with the cardiac crescent is considered to have an obligatory role in the induction of the cardiac fate. Various genetic and biochemical perturba- tions in several organisms have shown a key role for BMP members of the transforming growth factor β superfamily expressed in endoderm as well as in adjacent ectoderm and extraembry- onic tissues in specifying and/or maintaining the myocardial lineage.
Studies from the xenopus and chick models suggested that cardiogenesis is inhibited by Wnt-mediated signals from the underlying neu- ral tube activating the canonical Wnt pathway.
Based on the same animal models, cardiac dif- ferentiation was induced by Wnt-binding pro- teins (crescent and Dkk-1) secreted from the anterior endoderm. However, two recent articles suggest that the role of the Wnt family of pro- teins in cardiomyogenesis is much more com- plex. Pandur et al.43demonstrated that Wnt-11, an activator of the noncanonical Wnt/JNK path- way, is required for cardiogenesis using the xenopus model and the pluripotent mouse embryonic carcinoma stem cell line P19.
Nakamura et al.,44using the P19 cell line as well, revealed that the canonical β-catenin pathway of Wnt signaling is actually activated very early during mammalian cardiogenesis.
In response to the inductive signal, the car- diac crescent activates several transcriptional regulators of the cardiac program including Gata4/Gata5/Gata6, Nkx 2-5, myocyte enhancer factor (Mef2b/Mef2c), and T-Box 5/20 – and a positive cardiac cross regulatory network is established.45 A powerful transcription factor termed myocardin was recently identified and
shown to coactivate transcription of several car- diac-specific gene promoters in conjunction with serum response factor.46
Possible strategies for increasing the cardiomy- ocyte yield during hESC differentiation may thus include the use of different growth factors, overexpression of cardiac-specific transcrip- tion factors, coculturing with feeder layers, and mechanical factors. Directed differentiation of ESCs to the cardiac lineage in the murine model was achieved using a variety of soluble factors including dimethylsulfoxide,47retinoic acid,48and more recently BMP-2 and transforming growth factor β,49and ascorbic acid.50Xu et al.51demon- strated enhancement of cardiac differentiation in the hESC model by using 5-aza-2′-deoxycytidine but surprisingly not by dimethylsulfoxide or retinoic acid. There is also evidence to suggest that lessons learned from early cardiac differentiation in the model systems, described above, may also be applicable to the hESC.52 The cardiogenic inductive role of the primitive visceral endoderm was also demonstrated to have a role in the car- diomyocyte differentiation of the hESC line in an elegant study conducted by Mummery et al.27 Co-culturing of an hESC line (hES2) that does not regularly differentiate spontaneously to cardio- myocytes, with END-2 cells (a visceral endoderm- like cell line) provided the missing trigger for cardiac differentiation.27
Myocardial Regeneration Strategies Using hESCs
It is generally believed that adult cardiomyocytes have limited regenerative capacity and that this ability is insufficient to overcome the massive heart cell loss that occurs, for example, during an extensive myocardial infarction leading to the development of progressive heart failure.
Congestive heart failure is a growing epidemic in the western world because of the increasing age of the population and the improved postin- farction survival rates. Cardiac transplantation is currently the treatment of choice for end- stage heart failure. However, with the number of available donor organs limiting this treatment to only a minority of the patients, the develop- ment of new therapeutic paradigms for heart failure has become imperative. Cell replacement therapy is emerging as an innovative therapeutic approach for the treatment of degenerative
heart diseases. This therapeutic approach is based on the assumption that myocardial func- tion may be improved by repopulating diseased areas with a new pool of functional cells. Based on this assumption, a number of myogenic cells have been suggested for tissue grafting includ- ing skeletal myoblasts53; fetal,54,55 neonatal,56,57 and adult56 cardiomyocytes; smooth muscle cells58; murine embryonic stem cells59–62; and hematopoietic and mesenchymal bone marrow- derived stem cells.63
The ideal donor cell should exhibit the elec- trophysiological, structural, and contractile properties of cardiomyocytes and should be able to integrate structurally and functionally with host tissue. In addition, it has to retain an initial high proliferative potential that may enable improved colonization of the scar tissue. The ability to undergo genetic manipulation ex vivo in order to promote desirable characteristics such as resistance to ischemia and apoptosis and improved contractile functions may be another advantage of such an ideal cell type. Finally, the optimal candidate cell should have an autolo- gous origin or retain minimal immunogenicity and should be readily available in large quanti- ties for transplantation. Unfortunately, none of the currently available candidate cell sources exhibit all of the aforementioned properties.
The capability of bone marrow-derived stem cells to regenerate the heart by transdifferentia- tion to cardiomyocytes63 has been challenged by several studies demonstrating cell fusion of the progenitor cells with the host cells.64,65 Moreover, recent studies suggest that the bone marrow-derived stem cells continue to differen- tiate along the hematopoietic lineage66,67and the possible left ventricle functional improvement noted may not be related to transdifferentiation into the cardiac lineage but rather may result from indirect mechanisms such as their ability to enhance blood vessel formation.68
The autologous origin and the high availabil- ity of adult skeletal muscle cells led to their application in recently conducted phase I clini- cal trials.69However, although previous reports suggest that these cell may have the ability to adopt cardiac-like phenotype after cardiac transplantation, it is now clear that they do not possess such a capability.53,70Moreover, the rel- atively high rate of ventricular arrhythmias observed in the initial phase I clinical trials may stem from the differences in the electrophysio- logical properties between the host cells and the
engrafted myotubes and may further limit this approach.
Although a number of cell sources have been used in the aforementioned studies, the inher- ent electrophysiological, structural, and con- tractile properties of cardiomyocytes strongly suggest that they may be the ideal donor cell type. The clinical utility of fetal and neonatal cardiomyocytes is hampered, however, by the inability to obtain human cardiomyocytes in sufficient numbers because of practical and eth- ical reasons. Nevertheless, animal studies using fetal and neonatal cardiomyocytes have been instrumental to establish the proof-of-concept for the possible role of cell-based myocardial repopulation in improving myocardial per- formance of the injured heart. The fundamental work by Soonpaa et al.55demonstrated the abil- ity of fetal cardiomyocytes transplanted into mice hearts to survive, align, and form interca- lated disks with host cells. Further studies in animal models of myocardial infarction demonstrated that grafting of cardiomyocytes from fetal and neonatal sources was associated with smaller infarcts,71 prevented cardiac dilatation and remodeling,72and also improved ventricular function.73The mechanisms under- lying these functional improvements may be multifactorial and may include a direct contri- bution to contractility by the transplanted cells, attenuation of the remodeling process by changing the architectural and structural prop- erties of the scar, and improvement in the func- tion of viable tissue within the border zone by induction of angiogenesis.
The derivation of the hESC lines offers a num- ber of potential advantages over the currently available candidate donor cells. hESC are cur- rently the only cell source that can potentially provide, ex vivo, an unlimited number of human cardiac cells for transplantation. Second, the abil- ity of the ESCs to differentiate into a plurality of cell lineages may be utilized for transplantation of different cell types such as endothelial progenitor cells for induction of angiogenesis, and even spe- cialized cardiomyocytes subtypes (pacemaking cells, atrial, ventricular, etc.) tailored for specific applications. Third, because of their clonal origin, the ESC-derived cardiomyocytes could lend themselves to extensive characterization and genetic manipulation to promote desirable char- acteristics such as resistance to ischemia and apoptosis, improved contractile function, and specific electrophysiological properties. Fourth,
the embryonic stem-derived cells could also serve as a platform and a cellular vehicle for different gene therapy procedures aiming to manipulate the local myocardial environment by local secre- tion of growth promoting factors, various drugs, and angiogenic growth factors. Last, the ability to generate potentially unlimited numbers of car- diomyocytes ex vivo from the hESC may also bring a unique value to tissue engineering approaches.
Although hESDCMs could theoretically have the potential to fulfill most of the properties of the ideal donor cell, a number of critical obsta- cles need to be overcome before clinical appli- cation: (1) Studies assessing the ability of the cells to survive and integrate upon transplanta- tion to the normal and diseased myocardial host tissue should be conducted. (2) Strategies need to be developed for directing hESC differ- entiation into the cardiac lineage (as discussed above). (3) Purification of the cardiomyocyte population should be achieved using selection protocols. (4) Upscaling of the culturing tech- niques is needed to yield a clinically relevant number of cells for transplantation. (5) A trans- plantation technique should be developed to enable proper alignment of the graft tissue, high seeding rate of the transplanted cells, and min- imal damage to the host tissue. (6) Strategies aimed at preventing immunologic rejection of the cells should be developed.
Optimal functional improvement after cell grafting would require structural, electrophysio- logical, and mechanical coupling of donor cells to the existing network of host cardiomyocytes.
In a recent study, we tested the ability of the hESDCMs to integrate structurally and func- tionally with host cardiac tissue both in vitro and in vivo. Initially, the ability of the hESDCMs to form electromechanical connections with primary cardiac cultures was assessed in a high- resolution in vitro coculturing system. Primary cultures were created from neonatal rat ventric- ular myocytes. The contracting areas within the EBs were then mechanically dissected and added to the cocultures. Interestingly, within 24 hours postgrafting, we could detect synchronous con- traction in the cocultures that persisted for sev- eral weeks. Detailed studies of the hybrid cultures using the multielectrode array mapping technique demonstrated tight electrophysiologi- cal coupling between the two tissue types. The electrophysiological assessment was reinforced by demonstrating the development of gap junc-
tions between the human and the rat cells as suggested by Cx43 immunostainings.
To demonstrate the ability of the hESDCMs to survive, function, and integrate also in the in vivo heart, we assessed their ability to pace the heart and to function as a “biological pace- maker.” To examine this possibility, the cells were transplanted to the posterolateral region of the left ventricle in a swine model of slow hear rate. After cell grafting, a new ectopic ven- tricular rhythm was detected in 11 of 13 animals studies, in 6 of which it was characterized by sustained and long-term activity [Figure 3.4A (see color section)]. Pathological studies vali- dated the presence and integration of the grafted hESDCMs at the site of transplantation.
[Figure 3.4B (see color section)]. Three-dimen- sional electrophysiological mapping revealed that this ectopic ventricular rhythm originated from the area of cell transplantation (Figure 3.4C–E (see color section)]. Pathological studies validated the presence and integration of the
grafted hESDCMs at the site of transplantation [Figure 3.4D (see color section)].
Cell Selection Strategies
Although the development of directed differen- tiation systems is essential for increasing car- diomyocyte yield from the hESC, it is unlikely that the degree of purity that will be achieved would be sufficient for clinical purposes.
Because the beating EBs are comprise a mixed population of cells, selection strategies are cru- cial not only for increasing cardiomyocyte num- bers but also for preventing the presence of other cell derivatives as well as ensuring the absence of pluripotent stem cells carrying the risk of teratomas. In addition, a similar strategy may aid in selecting specific cardiac cell types.
An elegant selection scheme to generate pure cardiomyocyte populations was suggested by Klug et al.60in the mESC model. Undifferentiated Tissue
engineering
Growth
factors Electrophysiological charaterization Tolerance inducing strategies :
Somatic nuclear transfer.
Generation of a universal donor cell.
Hematopoetic chemerism. Human ES cell
lines
Pure Human ES cells
Derived CM
Differentiated and matured CM
Regeneration of The infracted
myocardium
Biological pacemaker
Drug Discovery
& Screening Growth factors promotingCardiogenesis
Genetic selection
Cell reprogramming
Hypoxia and apoptosis resistance Controlled Augmentation of cell proliferation Upscaling
Figure 3.3. Potential applications of human embryonic stem (ES) cell-derived cardiomyocytes. The diagram describes some of the potential appli- cations of hESDCMs as well as the possible obstacles to be overcome. CM, cardiomyocyte.
mESCs were transfected with a fusion gene coding resistance to neomycin under the regula- tion of the cardiac specific α-myosin heavy chain promoter. In addition, the transgene also carried sequence encoding hygromycin resistance enabling the selection of the stably transfected undifferentiated mESCs. Differentiation was then induced, and cardiomyocytes were selected based on the resistance for neomycin. A slightly differ- ent approach was reported by Muller et al.73a using GFP driven by the CMV enhancer and a ventricle specific promoter (MLC-2V). In con- junction with the use of Percoll gradient centrifu- gation and subsequent fluorescence-activated cell sorter, this strategy yielded a relatively pure (97%) population of ventricular cells. Although achieving high efficiency of stable transfection in the hESC has been difficult to achieve previously, the recent description of successful utilization of lentiviral vectors in these cell74,75 will foster the application of the selection strategies described above also in the hESC model.
Upscaling
The left ventricle of the human heart contains approximately 5.8 × 109cardiomyocytes.76Given the fact that 25% of the cells are lost during a typ- ical myocardial infarction leading to heart failure, cell transplantation strategies aiming to com- pletely regenerate the myocardium would have to use at least 109cells. Generation of this number of cells can be achieved by increasing the number of ESCs used for cardiomyocyte differentiation, by using directed differentiation systems and enrichment strategies, and by increasing the abil- ity of the cells to proliferate after cardiomyocyte differentiation. Zweigerdt et al.,77using the mESC model, combined the aforementioned cardiomy- ocyte selection strategies with the bioreactor technology to generate more than 109vital car- diomyocytes in a single 2-L bioreactor run.
Genetic and Tissue Engineering
Optimal regeneration of the infarcted myocardium leading to systolic augmentation would undoubtedly depend on the ability of the grafted tissue to form a functional syncytium with the host heart. Thus, any expected func- tional improvement would require the long- term survival of the grafted cells, the presence of
a critical tissue mass, and the appropriate align- ment and integration of the donor cells.
Increasing the survival of engrafted cells is a critical issue because the hostile ischemic scar tissue may result in significant cell death.78–80 Because adequate vascularization of the graft tissue is critical to its survival, the potential abil- ity to generate “smart hESDCMs” resistant to apoptosis and ischemia and capable of promot- ing graft vascularization by secretion of angio- genic growth factors may bring an added value to this. Future studies should also determine the ideal nature of the graft (e.g., individual cells, beating EBs, or combined with scaffolding bio- materials). Additional factors to be determined include the ideal degree of differentiation of the transplanted cells and the appropriate delivery method such as epicardial injections, transendo- cardial, or via the coronary circulation.
The ability to generate potentially unlimited numbers of cardiomyocytes ex vivo from the hESC may also bring a unique value to tissue engineering approaches. This new discipline combines functional cells with three-dimensional polymeric scaffolds to create tissue substitutes.
The possible advantages of this approach versus direct cell transplantation may lie in the ability to control graft shape and size, to control the align- ment of the engrafted cells, and to avoid the unpreventable initial cell loss encountered with the latter approach.81
Immunogenicity of the hESCs and Strategies Aimed at Achieving Tolerance
A major obstacle for the utilization of hESC derivatives in regeneration of different tissue types is the prevention of their immune rejec- tion. Initial characterization of the immuno- genicity of the hESC was conducted by Drukker et al.82The hESCs were shown to express a rela- tively low level of human leukocyte antigens (HLA) class I molecules. This expression was only moderately increased after differentiation in vitro (to EBs) and in vivo (to teratoma cells) but was significantly augmented after inter- feron-γ treatment. No expression of HLA class II molecules and the ligands for NK cell receptors was detected on the hESCs or their differenti- ated products. However, this study did not examine in detail the possible effects of the degree of EB maturation and differentiation and the heterogeneity of the cells within the EB on
the immunogenicity of the cells. In addition, possible expression of HLA class II molecules should also be assessed in vivo especially in the setting of an inflammatory response such as occurs during graft rejection.
Several strategies aimed at achieving immunologic tolerance were suggested. One of the most promising strategies is based on the generation of isogenic hESC lines tailored specifically for each patient. Recently, Hwang et al.83demonstrated that, using somatic nuclear transfer technology, this strategy may become technically possible. The authors derived a hESC line from an enucleated oocyte after somatic nuclear transfer. Although nuclear transfer may be a promising strategy, it might be limited by technical constraints as well as by ethical reasons. Another attractive strategy for induc- ing tolerance is hematopoietic chimerism.
Theoretically, hematopoietic chimerism may be achieved by transplanting hematopoietic stem cells derived from hESCs. After cell engraftment, the host will obtain tolerance because of the neg- ative selection of alloreactive T cells in the thy- mus. Hence, various differentiated derivative of the specific hESC line could be then safely trans- planted without the risk for immune rejection.
An alternative strategy might be to establish
“banks” of major histocompatibility complex antigen typed hESCs. The optimal solution for prevention of immune rejection may be the gen- eration of a universal donor hESC line. These could be achieved by silencing genes associated with the assembly or transcriptional regulation of the major histocompatibility complexes or by overexpression of FAS ligand in the hESCs and thereby inducing apoptosis of the infiltrating T lymphocytes mediating immune rejection.
Summary and Future Potential Research Directions
The derivation of the hESC lines and the resulting cardiomyocyte differentiation system may bring a unique value to several basic and applied research fields. Research based on the cells may help to elucidate the mechanisms involved in early human cardiac lineage commitment, differ- entiation, and maturation. Moreover, this research may promote the discovery of novel growth and transcriptional factors using gene trapping techniques, functional genomics, and
proteomics as well as providing a novel in vitro model for drug development and testing. Finally, the ability to generate, in vitro for the first time, human cardiac tissue provides an exciting and promising cell source for the emerging discipline of regenerative medicine and myocardial repair.
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