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Cardiovascular Research 2003 58(2):313-323; doi:10.1016/S0008-6363(03)00264-5
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Copyright © 2003, European Society of Cardiology

Human embryonic stem cells for cardiovascular repair

Sharon Gerecht Nira,b, Robert Davidc, Marc Zarubac, Wolfgang-M. Franzc and Joseph Itskovitz-Eldorb,d,*

aBiotechnology Interdisciplinary Unit, Technion—Israel Institute of Technology, Haifa, Israel
bDepartment of Obstetrics and Gynecology, Rambam Medical Center, P.O. Box 9602, Haifa 31096, Israel
cMedical Department I, Ludwig-Maximilians University, Klinikum Grosshadern, Munich, Germany
dFaculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel

itskovitz{at}rambam.health.gov.il

* Corresponding author. Tel.: +972-4-854-2536; fax: +972-4-854-2503.

Received 17 December 2002; accepted 29 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
The critical loss of functional cardiomyocytes causes severe deterioration of pump function, resulting in heart failure. The possibility to regenerate or repair damaged or ischemic cardiac tissue is a great challenge for the future treatment of end-stage heart failure. As cardiomyocytes cannot be regenerated in adults, current therapeutic modalities for the treatment of end-stage heart failure are limited and include medical therapy, mechanical left ventricular assist devices, and cardiac transplantation. This review will focus on the potential use of human embryonic stem (hES) cell-derived cardiomyocytes and vascular cells, as a therapeutic tool for the treatment of myocardial infarction and end-stage heart failure.

KEYWORDS Ischemia; Stem cells; Developmental biology; Myocytes; Angiogenesis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
Cardiovascular diseases are the most frequent cause of death in the western world. In the United States, congestive heart failure—the ineffective pumping of the heart caused by the loss or dysfunction of heart muscle cells—afflicts 4.8 million people, with 400,000 new cases each year. The main contributor to the development of this condition is myocardial infarction (MI), affecting nearly 1.1 million Americans each year. End stage heart-failure still has a limited prognosis underlined by the fact that only 50% of these patients survive the following year [1].

Cardiac transplantation is the treatment of choice for end-stage heart failure. However, its application is limited by the availability of donor organs. Another major obstacle is the immune response, which requires life long immunosuppressive therapy. Even in the case of successful transplantation, frequent failure of donor organs mainly due to transplantation vasculopathy remains still unsolved [2]. Regeneration or repair of damaged or ischemic cardiac may be achieved using cell therapy referring the transplantation of healthy, functional and propagating cells to restore the viability or function of deficient tissues. Stem cells are fundamentally characterized by prolonged self-renewal and long-term potential to form differentiated cell types. In most adult tissues, stem or progenitor cells are mobilized in response to environmental stimuli. But stem cells present in adult tissues form only a limited number of cell types. In early mammalian embryos at the blastocyst stage, the inner cell mass (ICM) is pluripotent. Therefore, the identification, derivation and characterization of human embryonic stem (hES) cells, may open the door to the rapidly progressing field of therapeutic cell transplantation.


   2 Development of the circulatory system
Top
 Abstract
 1 Introduction
 2 Development of the...
3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
Consisting of a heart, blood cells, and an intricate system of blood vessels, the circulatory system provides nourishment to the developing human embryo. It is the first functional unit in the developing embryo, while the heart is the first functional organ [3]. The heart primordium forms within a cardiogenic area located cranial and lateral to the brain. On day 19, angioblasts in the splanchnopleuric mesoderm respond to inductive signals from the endoderm to form lateral endocardial tubes, and then during embryonic folding in the fourth week, these vessels are translocated to the thoracic region, where they fuse to form the primitive heart tube. From week 5 to 8, the primitive heart tube undergoes folding, remodelling, and septation to form the four-chambered heart. Sinistral looping positions the regions of the heart that will form the primitive atria and ventricles and raises the inflow vessels to the level of the outflow tracts [4]. Thus, although the heart is the first functional organ of the body, it does not begin to pump until the vascular system of the embryo has established its first circulatory loops. Rather than sprouting from the heart, blood vessels form independently, linking up to the heart soon afterwards. In human embryos, the first blood vessels form within the yolk sac mesoderm in conjugation with blood cells on day 18 [4]. In the latter process, referred to as vasculogenesis, mesodermal angioblastic cysts fuse to form networks of angioblastic cords that expand, coalesce and invade embryonic tissues [4] to create the arterial, venous, and lymphatic channels. Blood vessels are also constructed by another process called angiogenesis which refers to the sprouting, remodelling and spreading of the primary network into a distinct capillary bed, arteries, and veins [6]. It is important to realize that the capillary networks of each organ arise within the organ itself, and are not extensions from larger vessels [3].


   3 Transplantation of non-embryonic stem cells
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
Remodelling caused by myocardial infarction is a common cause of ventricular dilatation and heart failure [7]. In the course of remodelling, necrotic cardiomyocytes are lost, a process which is accompanied by the formation of granulation tissue. Simultaneously, neovascularization in the peri-infarct area takes place. The latter is required for the survival of surrounding hypertrophic cardiomyocytes, and is meant to prevent further loss of cardiomyocytes caused by apoptosis [8]. Finally, the remodelling process leads to a formation of fibrous scar tissue, which is non-contractile and may expand, causing further cardiac impairment and heart failure [7]. Improvement of the myocardial function has been achieved in experimental animal models of heart failure and infarction, transplanting exogenous adult and embryonic cell types into damaged myocardium, which have been useful in reducing ventricular remodelling via the introduction of myocytes and improvement of the vascular supply. The transplanted cells included embryonic, fetal and neonatal cardiomyocytes from rodents and pigs [9–13] as well as human adult and fetal cardiomyocytes [14,15], autologous adult atrial cells [16], and dermal fibroblasts [17]. Independently of the cell type used, the improved myocardial function accompanying the transplantation may either be caused by the ability of the muscle cells to contract or the passive contribution of mechanical vigour to the myocardial architecture. In addition, angiogenesis can be induced in host scar tissue.

Most important factors limiting the future use of human fetal cardiomyocytes as donors for cardiac repair are ethical issues as well as limited supply. In addition, it is not clear whether intra-cardiac grafts derived from fetal cardiomyocytes can integrate tissue-specifically and functionally into the host myocardium. Intra-cardiac grafts with these cells remained isolated and were unable to differentiate into adult cardiomyocytes [11]. A recent clinical study showed the successful transplantation of autologous skeletal muscle cells into myocardial scar tissue during bypass surgery leading to increased wall thickness, improved contractility as well as vascularisation [18]. However, 4 out of 10 patients developed severe arrhythmias requiring implantation of automated cardioverter defibrillators. This may be due to an ectopic electrical activity of the transplanted skeletal muscle cells, which do not integrate into the host myocardium via cellular contacts.

Recent advances in stem cell research now offer an alternative cell source for the treatment of heart failure by transplanting exogenous adult and embryonic stem cells. The past years have aimed at gaining donor cells such as skeletal myoblasts [19–21], adult stem cells [22–31] and embryonic stem cells [32–35] for repair of cardiac tissue.


   4 Embryonic stem cells as a source of cardiomyocytes for transplantation
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
Pluripotent stem cells were obtained from two embryonic sources: ES cells which were derived from the inner cell mass of the blastocyst, while embryonic germ (EG) cells were isolated from the germ streak giving rise to primordial germ cells [36–40]. These undifferentiated cell lines were found to be pluripotent, i.e. have the ability to differentiate into cells of all three primary germ layers in vitro [41]. This remarkable potential of pluripotency allowing the differentiation of approximately 210 different cell types is meaningful for the prospective use of ES cells in tissue engineering. In the cardiovascular field, ES cells were spontaneously differentiated into cardiomyocytes [42–46], endothelial and vascular smooth muscle cells [33]. In addition, ES cells unleashed the opportunity to study the development of heart muscle cells from very early cardiac precursors to adult cardiac cells. Methods for the differentiation and phenotypic characteristics of murine ES (mES)-derived cardiomyocytes have been established and defined [40,42]. mES cells remain in the undifferentiated state when grown on feeder cells of mouse embryonic fibroblasts, and/or treated with leukaemia inhibitory factor (LIF). They differentiated into cellular aggregates, called embryoid bodies (EBs), after withdrawal of LIF and removal from the feeder layer [36]. It has been shown that cells within the EB differentiate into a wide range of cardiomyocytic cell types, including atrial, ventricular, Purkinje, sinus nodal and pacemaker-like cells. These different phenotypes showed corresponding expression of cardiac-specific marker genes such as ion channels, structural proteins and receptors [40,42]. They could be identified based on their typical action potentials [40] and their pharmacological properties. mES cell-derived cardiomyocytes were easily detectable in the heterogenous cell population of the EB by their spontaneous beating activity. Because less than 10% of the whole cell population of an EB are cardiomyocytes, the selection of enriched cultures of cardiomyocytes has required genetic manipulation [32,47]. Pure cardiomyocytic cultures have been achieved by stably transfecting mES cells with plasmid bearing the neomycin resistance gene under control of the cardiac specific {alpha}-MHC-promoter [40]. Selection with G418 resulted in beating cardiomyocytes.

Klug et al. transplanted these genetically manipulated mES-derived cardiomyocytes into the left ventricular wall of dystrophic mdx mice [32]. After transplantation, the donor cells were found to be associated with the host cardiomyocytes. The G418-selected donor cardiomyocytes showed a differentiated cardiomyocytic phenotype, and were positive for myofibrillar proteins, MHC and the intermediate filament desmin. In ultrastructural analyses, the presence of intercalated discs was shown. This early study on grafting has meanwhile been transferred to rats bearing an experimentally induced myocardial infarction [34]. Cardiomyocytes were obtained from the mES cell line ES-D3 and transfected with the cDNA coding for green fluorescent protein (GFP). After the intramyocardial engraftment, GFP-positive ES-derived cardiomyocytes survived in the infarcted rat myocardium and ameliorated heart function [34]. The viability of the donor cells was proven by their positive immunostaining for sarcomeric {alpha}-actin, which is not expressed in adult rat myocardium. The subsequent differentiation into a mature cardiomyocytic phenotype was shown by expression of {alpha}-MHC and cardiac troponin I.

In a recent study of the same group, the effects of engrafted early-differentiated cells (EDCs) from mES cells transfected with vascular endothelial growth factor (VEGF) on cardiac function in postinfarcted mice were assessed. mES cells were co-transfected with GFP cDNA and transplanted into infarcted myocardium. Compared with the infarcted mice receiving cell-free medium, cardiac function significantly improved in the infarcted mES treated mice 6 weeks after transplantation. Moreover, there was an even greater improvement of heart function in mice implanted with EDCs over-expressing VEGF in comparison to EDCs alone. Frozen sections of infarcted myocardium with EDCs or EDCs-VEGF transplantation showed GFP-positive tissue. Transplantation of EDCs or EDCs-VEGF significantly increased the number of blood vessels in the MI area. However, the density of capillaries was significantly higher in the EDCs-VEGF animals than in the EDC mice. Double staining for GFP and connexin-43 indicating the formation of an electrophysiologically functional syncytium was positive in injured myocardium with EDC transplantation [35].

Pure endothelial culture had been achieved by establishing mES cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter, tie-1. The resultant purified endothelial cells were shown to incorporate into the neovasculature of transplanted tumors in nude mice [48]. Yamashita and colleagues injected endothelial progenitors derived from mES cells intracardially into a developing chick embryo [33]. This population was incorporated as endothelial and mural cells and contributed to the developing embryo's vasculature. Pure endothelial cells sorted from hES cells showed the capability to build microvessels within 3D scaffold transplant subcutaneously [49].


   5 Specific selection of ventricular cardiomyocytes from ES cells
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
In cardiac transplantation studies using ES cell-derived cardiomyocytes, mixed populations of cardiac cell types derived from EBs were grafted [32,34]. This would most probably induce arrhythmogenicity and impair long-term cardiac function. Therefore, a prerequisite for the future use of ES cell-derived cardiomyocytes in the treatment of chronic heart failure or myocardial infarction would be the specific isolation of ventricular cardiomyocytes from embryoid bodies. In order to receive a highly purified population for cellular transplantation, our group established a purification protocol based on the ventricular specificity of the 2.1 kb myosin light chain-2 (MLC-2v) promoter fragment which had been previously shown in transgenic mice and in a recombinant adenoviral vector system [50–52]. In four independent transgenic mouse lines, luciferase expression under control of the 2.1 kb-MLC-2v promoter was exclusively observed in the ventricular myocardium. The experiments indicated that the 2.1-kb fragment of the promoter contains all regulatory elements required for specific gene expression in differentiated ventricular cardiomyocytes. Following another approach and to establish tissue-specific adenoviral vectors, 2.1 kb MLC-2v/luciferase cassette was used for ventricular cardiomyocyte-specific gene expression. It was shown that in the adenoviral system the 2.1 kb-MLC-2v promoter is not active in other tissues as opposed to the {alpha}-MHC-promoter [51]. The luciferase activity of heart muscle-specific adenoviruses was exclusively found in the ventricular myocardium [52]. These data were confirmed using additional adenoviruses via β-galactosidase-expression [53,54]. All these results showed that the 2.1-kb fragment of the MLC-2v promoter provides an excellent tool for the over-expression of distinct gene products in the ventricular myocardium.

Transferred to ES cells, this means that through marker gene expression, ventricular cardiomyocytes could be distinguished from other cell populations present within the embryoid body. However, the above-mentioned marker proteins, β-galactosidase and luciferase, were found unsuitable for the purification of transplantable cardiomyocytes as their detection inevitably leads to cell death. An alternative approach lies within the use of in vivo fluorescent proteins, such as enhanced green fluorescent protein (EGFP). Therefore, for the isolation of ventricular cardiomyocytes from cultivated murine ES cells, EGFP was cloned under the control of the well characterized 2.1 kb MLC-2v promoter. An enhanced fluorescence signal was achieved by introducing the CMV-enhancer-sequence (Fig. 1), thereby obtaining nine EGFP positive cell clones. Four of these clones showed a significantly high number of fluorescent cells within the spontaneously contracting areas of the EBs [47]. Immunostaining using specific antibodies against skeletal muscle, cardiomyocytic and atrial proteins revealed the first indications of a ventricular identity of the fluorescent cardiomyocytes. Electrophysiological analyses of beating fluorescent ES cells 21 days after induction of differentiation showed ventricular properties for over 80% of the contracting cardiomyocytes. Significantly, at this time point of differentiation which corresponds to day 5–6 post partum, approximately 6% of the fluorescent cells corresponded to an embryonic type of cardiomyocytes. However, the time point of selection of the MLC-2v-based selection was found crucial with respect to the endogenous expression of MLC2v: in vivo MLC2v became restricted to the ventricle post partum whereas it was also expressed in parts of the atrium and the inflow tract during embryonic and fetal stages [55]. By stimulation using isoproterenol and carbachol the typical electrophysiological behaviour of the fluorescent cardiomyocytes could be verified [47]. More than 80% of the fluorescent cells were identified as ventricular cardiomyocytes based on their long plateau phase and a very negative diastolic potential between –60 and –80 mV. Typically, carbachol administration did not influence the characteristics of the ventricular action potentials, whereas isoprenalin shortened the plateau phase and led to an increase in the frequency of the action potentials. The latter was reversible after the washout of the drug [47]. After the immunohistochemical, electrophysiological and pharmacological characterization, a purification method was established, shown in Fig. 2: initially some of the cells within the heterogeneous population of the embryoid bodies were positive for EGFP. After an enzymatic separation of the cells, a first three- to fourfold enrichment of the EGFP-positive cardiomyocytes was achieved via density gradient centrifugation. Cell sorting based on EGFP fluorescence subsequently led to a highly purified population consisting of 97–98% fluorescent cardiomyocytes [47]. The purified cardiomyocytes contained the typical contractile fibres and an intact sarcomeric structure [47]. This showed the ventricular identity of the cardiomyocytes after FACS sorting as well as the high specificity of the CMV-MLC-2v-promoter construct. In this respect, the newly established expression cassette is superior to the {alpha}-MHC- and the {alpha}-actin promoters, which are not specific for subtypes of cardiomyocytes [32,56]. In this context it is important to mention that the isolation of cardiomyocytes via EGFP fluorescence as well as antibiotic selection [32] leads to the expression of foreign proteins within the cells most probably causing immunological problems after transplantation. In addition, after FACS the ventricular cardiomyocytes lose their electrophysiological properties and their contractile potential most likely due to stress during the procedure. For this reason, alternative MLC-2v promoter-based approaches for the isolation of ventricular cardiomyocytes is one of our main focuses of research.


Figure 1
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  		Fig. 1 Strategy for the selection of cardiomyocytes from in vitro differentiated, green fluorescent ES-cells via EGFP expression under the control of MLC-2v promoter. A CMV-enhancer-element was introduced 5' to the promoter to increase the fluorescence.

 

Figure 2
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  		Fig. 2 Purification protocol for viable in vitro differentiated green fluorescent cardiomyocytes.

 

   6 Pluripotent stem cells for vasculo- and angiogenesis
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
Early in embryonic development, blood vessel formation occurs by a process referred to as vasculogenesis, in which endothelial cell precursors differentiate, expand and aggregate to form a network of primitive tubules [5]. Endothelial cells were found to play a main role in the organogenesis of the mouse embryo: they promote liver organogenesis [57], induce pancreas differentiation [58], and were found to trans-differentiate into cardiac muscle cells under specific conditions [59]. Another study showed the ability of endothelial progenitor cells, harvested from peripheral blood of sheep, to expand ex-vivo on decellularized porcine iliac vessels, resulting in functional neovessels [60]. Since pluripotent ES cells can expand without apparent limit [61–63], ES cell-derived cells can be created in virtually unlimited amounts, making the potential therapeutic implications limitless. These implications include induction of tissue vascularization by cell transplantation or tissue engineering of vascular grafts, and building functional human neovessels ex-vivo. In addition to potential clinical applications, purified human embryonic endothelial cells may prove important for studying early human development and differentiation of embryonic stem cells into various tissues, for pharmacological studies and genetically modified endothelial cells.

Angiogenesis can be defined as the sprouting of capillaries from existing vessels that leads to the formation of new microvessels in previously avascular tissues. The molecular basis of angiogenesis is easily characterized by viewing the process as a stepwise progression [5,64]. The initial vasodilatation of existing vessels is accompanied by an increase in permeability and degradation of surrounding matrices allowing activated and proliferating endothelial cells to migrate and form lumens [5,64,65]. Therefore, the endothelial cell is a major player in processes involved in angiogenesis, migration and proliferation, eventually leading to tube formation. Peri-endothelial cells are recruited to support the endothelial tube, providing maintenance and modulatory functions to the vessel. These cells are pericytes in the capillaries, smooth muscle cells in larger vessels and cardiac myocytes in the heart. In the adult, angiogenesis occurs in physiological conditions, such as in the female reproductive tract, but more commonly in various pathological conditions. Most appreciated are the formation of new blood vessels to nurture growing tumors (tumor angiogenesis) and the formation of collaterals in an ischemic heart or limb [66,67]. Although the complex occurrences leading to angiogenesis are not completely understood, considerable interest is presently centered on the inhibition of new vascular growth to treat the spread of cancer. There is a great need for a suitable in vitro model system to study antiangiogenesis agents. Watenberg et al. compared the kinetics of endothelial differentiation within mouse EBs, which were grown with different antiangiogenesis factors [68]. Hence, hES cells may serve as an in vitro research model to examine the molecular mechanisms involved in human angiogenesis. The role of VE-cad in tube formation was examined by studying its angiogenic blocking potential using a 3D sprouting model of differentiated hES cells. A monoclonal anti human VE-cad inhibited sprouting and network-like structures derived from hES cells. These findings stress the importance of this molecule in human vasculogenesis and angiogenesis as a potential angiogenesis inhibitor.


   7 Human embryonic stem cells
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
hES cells which are derived from human preimplantation embryos at the blastocyst stage [61–63] were demonstrated to fulfil all the criteria defining embryonic stem cells: immortality, capability to proliferate indefinitely in culture while maintaining the undifferentiated phenotype and the capacity to form derivatives of all three germ layers. Hence they may serve as a source of numerous types of differentiated cells. hES cells remain in the undifferentiated state when cultured on inactivated mouse embryonic fibroblasts (MEFs) [61,62], human fetal fibroblast and adult epithelial cells [69] or foreskin cells [70]. They can also be grown on matrigel or laminin with media conditioned by MEFs [71]. Undifferentiated hES cells are characterized by the expression of surface markers typical of undifferentiated primate ES cells, such as stage specific embryonic antigen 3 (SSEA), SSEA4, Tra-1-60, and Tra-1-81, expression of the embryonic transcription factor Oct-4, positive for alkaline phosphatase activity and high levels of telomerase activity. hES cells are generally differentiated in two major ways. The first is in an unmanipulated manner which involves the removal of undifferentiated cells from feeder layer and the spontaneous formation of embryo-like aggregates, or EBs, in suspension [72]. As in the mouse system, hEBs are composed of cellular derivatives of all three primary germ layers of endodermal, ectodermal and mesodermal origin and some form large lumen, i.e. cystic EBs. Both cardiomyocytes and endothelial cells have been shown to arise during spontaneous hEB formation [49,73]. The second differentiation method is a manipulative one, which at some point involves disaggregation and/or culture with specific cytokines, resulting in purity or increase in proportion of specific cell types. Induction and enrichment of cardiomyocyte differentiation from hES cells can be achieved by both cytokine supplementation and density separation [74].


   8 Cardiomyocytes derived from hES cells
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 
In hES cells, a number of signal factors have been shown to induce differentiation into specific subtypes of cells or tissues. Specific stimuli for cardiac differentiation of hES cells are hepatocyte growth factor (HGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor β1 (TGFβ1) and retinoic acid [74]. Beating cardiomyocytes were shown to arise from spontaneous differentiated hES cells [72]. These spontaneously contracting areas appeared in 8.1% of the EBs and displayed structural and functional properties of early-stage cardiomyocytes [73]. In a recent publication, high-resolution activation maps using microelectrode arrays (MEA) demonstrated the presence of a functional syncytium with stable focal activation and conduction properties [75]. Although these studies used a single-cell clonal line, the H9.2 [73,75], cardiomyocyte differentiation in multiple parental hES lines, including H1, H7, H9 and the additional clonal line H9.1 [76] and in I3 and I6 (unpublished data) has also been shown. In this report, cardiomyocyte differentiation was enhanced when hES cells were treated with 5-aza-2'-deoxycytidine but surprisingly not retinoic acid as opposed to murine ES-cells and the previous reports on hES cells [74,75]. Furthermore, the differentiated cultures could be dissociated and a first enrichment of hES cell derived cardiomyocytes via Percoll gradient density centrifugation was achieved leading to a population of 70% pure cardiomyocytes. The enriched cells were proliferative and showed appropriate expression of cardiomyocytic markers. Fig. 3 summarizes the existing protocols for the generation and enrichment of cardiomyocytes from hES cells. However, so far the ultrastructural maturation of hES derived cardiomyocytes has not been shown to parallel that of adult cardiomyocytes as they display an early phenotype [73]. Biological differences seem to exist between murine and hES cells [75] and further studies are necessary to prove the ability to use hES-derived cardiomyocytes for cellular engrafting.


Figure 3
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  		Fig. 3 Protocols for cardiomyocyte derivation from hES cells.

 

   9 Vascular progenitors derived from hES cells
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
10 Challenges for the...
 Acknowledgments
 References
 
Blood vessels are generally comprised of two cell types, each serving a different function: internal endothelial cell lines form the channels for blood conduction, whereas external smooth muscle cells protect the fragile channels from rupture and control blood flow [64].

While the nature of endothelial precursors is not yet fully understood, it is evident that hematopoietic development and the generation of vascular smooth muscle cells (SMC) are tightly linked to vascular development. In the developing embryo, endothelial cells arise either from precursors which can produce endothelial cells (angioblasts), or from progenitors that give rise to both endothelial and blood cells (haemangioblasts) [64]. Blood cells appear first within the yolk sac and later within the embryo. The second hematopoietic emergence takes place separately within the embryo in the para-aorta-splanchnopleura region, which later contributes to the future aorta, gonads and mesonephros, i.e. the AGM region. Blood-forming activity in the AGM region has been demonstrated and assessed functionally in bird, mouse and human embryos [77]. Thus, hematopoietic progenitors emerge in early development in close physical association with endothelial cells, either in the extra-embryonic yolk sac or within the embryo. It seems that both hematopoietic and endothelial cells are derived from blast cell colonies generated from mouse ES cell-derived EBs [78]. In contrast, Nishikawa and colleagues have suggested that VE-cadherin+ cells (i.e. endothelial cells) in the embryonic mouse may produce hematopoietic cells [79–81]. In addition, early periendothelial SMCs associated with embryonic endothelial tubes have been shown to trans-differentiate from the endothelium, up-regulating markers of smooth muscle cell (SMC) phenotype (both surface markers and morphology) [82], and even a common embryonic vascular progenitor was later reported to differentiate into endothelial and SMCs [33].

The ability of hES cells to differentiate and form blood vessels can be easily observed during teratoma formation. Using HLA antigen, the formation of small human blood vessels containing both human and mouse-originating blood cells was noticed.

Since cystic mouse EBs have been documented for endothelial cell differentiation and primitive vasculature formation [83], it was highly desirable to apply it to differentiation of hES cells to vascular structures. Levenberg et al. were the first to show the formation of vascular network formation in developing EBs [49]. Following the kinetics of blood vessel formation in growing EBs, it has been noticed that intensive and complicated vascular-like channels can be observed in 10- and 13-day-old EBs [49]. Further examination showed that the initiation of vessels’ voids and network structures occurs in 1-week-old EBs while more mature structures can be observed in 1-month-old EBs. Gene expression kinetics indicated the existence of differences between mES and hES cells [49]. For example, high expression levels of vascular endothelial growth factor-A (VEGF) receptor 2 (VEGFR2) also known as fetal liver kinase1 (Flk-1), and AC133 appeared in undifferentiated hES cells. Flk-1 expression in undifferentiated hES cells was also reported by Kaufman et al. [84] but reported not to be expressed in undifferentiated mES cells [85,86]. Furthermore, Flk-1, an important receptor in mouse embryo development [87], was hardly changed during the first days of hES differentiation, suggesting that other markers may take part in human vascular development.

A simple method was developed for the enrichment of early endothelial progenitors derived from hES cells. Nishikawa et al. previously reported on a 2D differentiation system using type IV collagen as suitable for proximal lateral mesoderm of mES cells [80]. hES cell aggregates seeded on type IV collagen differentiated in the edges of colonies but remained in an undifferentiated state in the dense centre regions. This culture expressed all VEGF isomers, and Tie2, Flk-1, Ang2, and CD31-specific markers of endothelial cells. Some of the differentiated cells formed muscle/vascular arrangements, which were found to express smooth muscle {alpha}-actin (SMA). For the induction of a more defined population, individual hES cell-suspension was seeded on type IV collagen. Examination of the differentiated population revealed a high percentage of specific endothelial markers such as vascular endothelial cadherin (VE-cad), CD31 and Flk-1, which also expressed other endothelial/hematopoietic markers such as Tie2, Tal1, Gata2 and AC133 (Fig. 4). Administration with hVEGF matured these cells into endothelial cells producing von Willebrand factor (vWF) and with high lipoprotein metabolism. Up-regulation of the expression of SMC markers was achieved once supplemented with human platelet-derived growth factor BB (hPDGF-BB). In addition, colony formation unit assays revealed the ability to form different hematopoietic colonies. Vessel-like structures were formed once seeding the differentiated cells within 3D collagen and matrigel gels. Electron microscopy examination showed typical endothelial cell features and network structure with lumen (Fig. 5).


Figure 4
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  		Fig. 4 Progenitors molecular characterization. Specific endothelial and hematopoietic markers were expressed in progenitor cells but not in undifferentiating hES cells.

 

Figure 5
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  		Fig. 5 Electron microscopy of endothelial cells. HES cell-derived vascular progenitors seeded on matrigel formed typical vessel-like structures containing lumen, indicating their ability to differentiate into functional endothelial cells.

 

   10 Challenges for the future
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
Acknowledgments
 References
 
The primary therapeutic aim of the research on hES cells is to derive cells for the replacement of diseased or damaged tissue. Transplantation opportunities of cells derived from hES cells hold few obstacles [88]. Since hES cells spontaneously form teratomas in vivo [61,62], the first obstacle is their tumorigenic potential. Therefore, establishing a highly efficient purification protocol of the required cell type is crucial. Another concern is the potential of hES cells to transmit infections, but this can become marginal when using adequate derivation and culture standards. Another major objective will be to develop ways to overcome the immunological barriers [88,89]. Several approaches have been suggested to overcome this problem: (1) Minimizing the donor-recipient alloantigenic differences by either establishing a stem-cell bank or by ‘therapeutic cloning’ (genomic replacement); (2) Immunosuppressive therapy; (3) Genetic manipulation (to create a ‘universal-donor’); and (4) Induction of donor-specific tolerance (haematopoietic chimerism; donor-blood pre-transplantation; lymphocyte infusion peri-transplant).

A promising strategy to obtain cells for therapeutic use will have to fulfil several criteria such as the large-scale availability and proliferation of undifferentiated hES cells, as well as their large-scale differentiation. ES-cell derived cardiomyocytes terminate the cell cycle following the formation of multinuclei—a typical feature of terminally differentiated cardiomyocytes [40]. Therefore, the proliferative potential of ES-derived cardiomyocytes in vivo is probably limited and the availability of reasonable numbers of cardiomyocytes required to repair large myocardial infarctions in men may prove to be a major hindrance [10]. Furthermore, cardiac grafting of ES-derived cardiomyocytes so far led only to a modest reconstitution of the myocardium (7.3% of donor cells) in the left ventricle of infarcted rat hearts [34]. In vitro, however, yield could be increased by treatment with different reagents [75,90] and by the over-expression of zinc-finger transcription factor GATA-4 [91]. Whether these approaches can generate sufficient numbers of ES-derived cardiomyocytes for myocardial transplantation remains to be answered in the near future.

Time for primary review 28 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
References
 
We thank Hadas Perry for editing. The Fund for Medical Research and Development of Infrastructure and Health Services, Rambam Medical Center and the Technion Research and Development Foundation Ltd supported hES cell work. The work for the selection of ventricular cardiomyocytes from mES cells was funded by BMBF grant 01KV9560.


   References
Top
 Abstract
 1 Introduction
 2 Development of the...
 3 Transplantation of non...
 4 Embryonic stem cells...
 5 Specific selection of...
 6 Pluripotent stem cells...
 7 Human embryonic stem...
 8 Cardiomyocytes derived from...
 9 Vascular progenitors derived...
 10 Challenges for the...
 Acknowledgments
 References
 

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