The heart is the first organ to form during embryonic development. In 1965, by the use of fate mapping techniques, DeHaan et al. (1964, 1965) demonstrated the presence of mesodermal cells, which are committed to the heart by the early gastrula stage. In the mouse, the generation of a single heart tube is initiated between E7.5 and E (embryonic day) 8, and the first contraction of this primitive structure can be observed from E8.5–E9 (Fishman and Chien, 1997). In humans, endodermal inductive signals direct angioblasts located in the splanchnopleuric mesoderm to form lateral endocardial tubes. During embryonic folding in the fourth week, these vessels are translocated to the thoracic region, where they fuse to form the primitive heart tubes (Larsen, 1998). During weeks 5–8, the primitive heart tube undergoes folding, remodeling, and septation to form the four-chambered heart. Hence, although significant attention has been paid to myocardial differentiation during early cardiac morphogenesis, it is important to realize that endothelial and endocardial development are also critical during heart formation.
The mechanisms that regulate condensation of the preheart cells are largely unknown. A considerable amount of data has been documented on the morphology and physiology of adult heart cells, but models for the studying of early cardiac development are scarce; the first studies were carried out on organ-cultured chick hearts (Sperelakis, 1982), precardiac areas of blastoderm (McLean et al., 1978), and cultured mammalian heart cells of embryonic and neonatal origin (Wollenberger, 1985). The findings of these works are limited, though, since cell properties and normal cardiogenesis are disrupted during cultivation (Maltsev et al., 1993).
ISOLATION AND DIFFERENTIATION OF ESCs
Embryonic stem cells (ESCs) are undifferentiated cells isolated from the inner cell mass (ICM) of the embryo. At the expanded blastocysts stage, embryos are plated, either intact or following immunosurgical isolation from the ICM, onto a feeder layer (Fig. 1). Following several days of culture, the ICM is disaggregated and replated onto fresh feeders. The newly derived cell mass has the potential to develop into a variety of cell lineages. This quality, referred to as pluripotency, is determined by the cells' 1) immortality—indefinite self-renewal in culture (featuring high levels of telomerase expression); 2) maintenance of undifferentiated phenotype and normal karyotype; 3) ability to develop into all three primary germ layer derivatives, namely, ectoderm, mesoderm, and endoderm, both in vitro and in vivo; and 4) clonality—each single cell acquires the above features. This remarkable potential of pluripotency allowing the differentiation of approximately 210 different cell types is meaningful for the prospective use of ESCs in future cell therapeutic applications. In 1981, the first ESC line was established directly from mouse blastocysts (Evans and Kaufman, 1981). Since then, human ESC (hESC) lines were derived from fresh or frozen human blastocysts (Thomson et al., 1998; Amit et al., 2000; Reubinoff et al., 2000; Amit and Itskovitz-Eldor, 2002). Cell lines and clones (Amit and Itskovitz-Eldor, 2002) were established from those ICMs, and they retain normal phenotype and karyotype even after long-term culture. The protocols for ESC derivation are relatively simple and have remained unchanged until today (Smith, 2001), which facilitates their generation for both research and therapeutic usages. ESCs are generally differentiated in vitro where the removal of undifferentiated cells from their feeder layer leads to the spontaneous adherent differentiation or formation of embryo-like aggregates in suspension, called embryoid bodies (EBs). EB spontaneous differentiation results in the generation of cellular derivates of all three primary germ layers. Another differentiation procedure involves the induction or direction of specific lineage differentiation. This may be achieved by culture or genetic manipulation of the undifferentiated ESCs. Manipulated differentiation can lead to the generation of a purified population or an increase in the proportion of a specific cell fate.
APPLICATIONS FOR CARDIOGENESIS
Murine ESCs (mESCs) were terminally differentiated into cardiogenic cells (Wobus et al., 1991; Maltsev et al., 1993, 1994; Miller-Hance et al., 1993). Differentiation of mESCs (Wobus et al., 1991) recapitulates the development of cardiomyocytes from very early (e.g., cardiac precursor) to terminally differentiated (e.g., artrial, ventricular, chamber progentors, sinus-nodal cell, Purkinje, pacemaker-like cells) (Miller-Hance et al., 1993; Maltsev et al., 1994; Hescheler et al., 1997; Wobus et al., 1997; Hidaka et al., 2003).
Recent studies preformed on rat infraction models showed improvement in cardiac function following undifferentiated mESC transplantation (Min et al., 2002, 2003). Genetic manipulation designed to direct cardiac differentiation involved selection of cells expressing the α cardiac myosin heavy chain (MHC) gene (Klug et al., 1996), isolation of cells expressing ventricular-specific 2.1-kb myosin light chain-2 (MLC2V) (Muller et al., 2000), and isolation of Nkx2.5 (Hidaka et al., 2003). In the first case, mESCs transfected with α cardiac MHC promoter were subjected to antibiotic selection during differentiation. The ventricular resultant cell culture exhibited clear cardiocmyocyte characteristics both in vitro and in vivo when implanted in the hearts of adult dystrophic mice (Klug et al., 1996). Another approach used the transfection of enhanced green fluorescent protein (EGFP) under the transcriptional control of MLC2V promoter to mark ventricular-like cardiomyocytes differentiated from mESCs. These cells were sorted and found to display vanticular-cardiomyocyte-specific characteristics. The same strategy was recently used to isolate chamber-specific cardiac lineages from differentiating mESCs (Hidaka et al., 2003). All together, these studies indicate that a relatively simple genetic manipulation can be used to select essentially pure cultures of different types of cardiomyocytes from differentiating mESCs.
hESCs currently require an embryonic fibroblast feeder layer (Thomson et al., 1998; Richards et al., 2002) or their conditioned medium (Xu et al., 2001) in order to be maintained in an undifferentiated state. When removed from the feeder layer and transferred to suspension culture, hESCs begin to form hEBs (Fig. 2). hEBs often progress through a series of stages commencing as simple, morula-like structures and eventually forming cavitated and cystic-like EBs between days 7 and 14 of postdifferentiation development (Itskovitz-Eldor et al., 2000; Odorico et al., 2001). During this spontaneous differentiation a minority of cystic hEBs display rhythmic pulsing (Itskovitz-Eldor et al., 2000; Kehat et al., 2001). The first pulsing of a hEB was timed at a synchronous rhythm of about 30 pulses per minute (Itskovitz-Eldor et al., 2000). Specific marker examination revealed that the central cavity of this hEB was surrounded by cardiac muscle cells marked with α-cardiac actin (Itskovitz-Eldor et al., 2000). Screening of growth factors for induced differentiation showed that the mesoderm is the most easily triggered during differentiation of hESCs (Schuldiner et al., 2000). Furthermore, myocardial-specific cell enrichment was accomplished by the addition of transforming growth factor (TGF)-β (Schuldiner et al., 2000). Detailed examination of hESC spontaneous differentiation into cardiomyocytes revealed that the spontaneously contracting areas appeared in 8.1% of the EBs (Kehat et al., 2001). The differentiation strategy included dissociating hES single-cell clone H9.2 (Amit et al., 2000) into small clumps of 3–20 cells, growing them in suspension for 7–10 days, and finally plating them on gelatin-coated culture dishes. Contracting areas with a diameter range of 0.2–2 mm appeared 4–20 days after plating in the outgrowth of the EB, and continued beating for up to five weeks (Kehat et al., 2001). Structural and functional examination revealed that the spontaneous contracting areas displayed properties of early-stage cardiomyocytes (Fig. 2). Our further examination revealed that the plating of mid-size cystic EBs generated from the H9.2 single-cell line can result in almost 30% of contacting EBs. Statistical analysis revealed that approximately seven days after plating the EBs, spontaneous contracting areas appear in 20% of the total contracting EBs. Within 15 days, spontaneous contraction will come into view in 90% of the total contracting EBs; i.e., most spontaneous contraction occurs within 23–25 days of differentiation. In a recent publication, morphological analysis revealed an isotropic tissue of early-stage cardiac phenotype, with gap junctions immunostained with connexin43 and connexin45 but not connexin40 (Kehat et al., 2002). High-resolution activation maps using microelectrode arrays (MEAs) demonstrated the presence of a functional syncytium with stable focal activation and conduction properties along the contracting area of hEBs (Kehat et al., 2002). Although these works used a single-cell clonal line, cardiomyocyte differentiation in multiple parental hESC lines, including H1, H7, H9, the additional clonal line H9.1 (Xu et al., 2002), and hESC lines derived in our laboratory, such as I3 and I6 (unpublished data), has also been shown. Xu et al. (2002) used a different protocol of four-day-old EBs from different cell lines and clones (H1, H7, H9, H9.1, and H9.2) that were seeded at low density. The effects of differentiation reagents dimethyl-sulfoxide (DMSO), all-trans retinoic acid (RA), or 5-aza-2′-deoxycytidine (5-aza-dC) were assessed at different times during differentiation. In addition, enrichment of cardiomyocytes using discontinuous Percoll gradient was carried out. DMSO and RA did not affect hESC cardiomyocyte differentiation, perhaps due to different signaling pathways in human and murine. Alternatively, the differentiation was significantly enhanced by treating the cells with the demethylation reagent 5-aza-dC or by using a Percoll gradient separation technique. By using their protocol, Xu et al. (2002) were able to reach spontaneous contracting cells in 70% of their EBs.
A different work showed that cariomocyte-induced differentiation occurs with co-cultures of hESCs and END-2 cells, a P19 embryonic carcinoma-derived cell line possessing the characteristics of visceral endoderm (Mummery et al., 2002). So far, this is the only procedure where the formation of hEBs is not a prerequisite for cardiomyocyte differentiation (Mummery et al., 2002).
APPLICATIONS OF VASCULO- AND ANGIOGENESIS
In the embryo, vasculogenesis is the process in which blood vessel formation occurs by differentiation of vascular endothelial cells from angioblastic precursors, which in turn give rise to a primitive vascular plexus (Risau, 1997; Yancopoulos et al., 2000). In contrast, angiogenesis refers to the process in which preexisting vessels sprout or split to form new vessels (Carmeliet, 2000).
Doetschman et al. (1985) were the first to report on hematopoietic cells surrounded by endothelial cells on the surface of cyctic EBs. Typical vascular networks that are connected by typical endothelial junctions and contain hematopoietic cells were further detected within cyctic EBs (Wang et al., 1992; Risau, 1997). mESC endothelial differentiation could also be observed using matrix adherent cultures, i.e., in the absence of EB formation. Endothelial differentiation could be observed using small mEBs (less than four days old) adherent to gelatin-coated dishes (Bautch et al., 1996) or mESCs seeded for differentiation on type IV coated dishes (Hirashima et al., 1999). A number of markers were identified for the early mouse vasculature, such as vascular endothelial growth factor-A (VEGF) receptors VEGFR2, also known as fetal liver kinase-1 (flk-1), and VEGFR1; vascular endothelial cadherin (VE-cad); Tie2; CD34; and SCL/TAL (Vittet et al., 1996; Drake et al., 1997; Drake and Fleming, 2000). However, platelet endothelial cell adhesion molecule-1 (PECAM1/CD31) has proven particularly useful due to its abundant early expression in vascular development (Vecchi et al., 1994). Endothelial cord formation was enhanced by the addition of free TGF-β, suggesting that during in vitro mESC differentiation, latent TGF-β-binding protein-1 (LTBP-1) regulates endothelial cell development and differentiation by modulating TGF-β formation (Gualandris et al., 2000). Efficient differentiation to endothelial cells could be observed while using VEGF-specific isomer 165 rather than 121 (Yamashita et al., 2000). Genetic manipulation to direct endothelial differentiation involves the establishment of mESC 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 (Marchetti et al., 2002). Using knockout analysis, other genes, such as VE-cad, Gata4, β1-integrins, etc., were shown to be crucial for normal vasculature formation during mESC differentiation (Feraud and Vittet, 2003).
Not long ago, conventional knowledge claimed that endothelial and vascular smooth muscle cells (v-SMCs) arise from separate precursor cells through rounds of cell division and specialization (Carmeliet and Collen, 1999; Gale and Yancopoulos, 1999). It was thought that after vasculogenesis, a later phase of vascular development occurred. This phase involved the sprouting and penetration of vessels into previously avascular regions of the embryo, and also the differential recruitment of associated supporting cells, such as SMCs and pericytes (Gale and Yancopoulos, 1999; Yancopoulos et al., 2000). However, in recent years, it has become evident that the generation of v-SMCs is tightly linked to vascular development. Early periendothelial SMCs associated with embryonic endothelial tubes have been shown to transdifferentiate from the endothelium, upregulating markers of SMC phenotype (both surface markers and morphology) (DeRuiter et al., 1997). Recently, mature vascular endothelium has been shown to give rise to SMCs via transitional cells, co-expressing both endothelial and SMC-specific markers (DeRuiter et al., 1997). Yamashita et al. (2000) discovered that Flk-1-positive cells derived from differentiated mESCs are common embryonic vascular progenitors that differentiate into endothelial and SMCs. SMCs arising from these progenitors expressed the atypical α-smooth muscle actin (a-SMA) marker, together with an entire set of SMC markers, and also surrounded endothelial channels when injected into chick embryos (Yamashita et al., 2000). A new work also showed that these progenitors are incorporated during tumor angiogenesis (Yurugi-Kobayashi et al., 2002).
With the attractive therapeutic potential of vasculogenic cells in medicine, much attention has been focused upon the differentiation of hESCs into mature vasculogenic lineages. Levenberg et al. (2002) preformed a kinetics analysis for the expression of specific endothelial markers during spontaneous hEB formation. The levels of endothelial markers PECAM1, VE-cad, and CD34 increased during the first week of hEB differentiation, reaching a maximum on days 13–15 and indicating a differentiation process toward endothelial cells. In contrast to mESCs, Flk-1 was shown to be expressed in undifferentiated hESCs (Kaufman et al., 2001; Levenberg et al., 2002) and increased very slightly during differentiation (Levenberg et al., 2002). However, on day 13 all hEBs had defined cell areas expressing PECAM1 arranged in vessel-like structures in correlation with VE-cad and von Willibrand factor (vWF) expression. PECAM1-positive cells (2%) were also sorted from 13-day-old EBs. The isolated PECAM1+ cells were shown to possess embryonic endothelial cell (EEC) features, such as acetylated low-density lipoprotein (ac-LDL) incorporation, formation of cord-like structures on Matrigel in vitro, and formation of microvessels containing mouse blood cells upon transplantation into SCID mice (Levenberg et al., 2002). Rather than selecting a progenitor from differentiating hEBs via specific cell markers, a method for inducing hESC mesoderm differentiation based on two-dimensional culture manipulations was established. The underlying assumption was based on the notion that neither co-culture nor EB formation is necessary for efficient differentiation of hESCs into vascular-lineage cells. Single-cell-suspension culture of hESCs on type IV collagen matrix in specific cell seeding concentration induced an enriched endothelial progenitor population. Exposing the culture to specific angiogenic mitogens such as VEGF and platelet-derived growth factor BB (PDGF-BB) resulted in endothelial cells and v-SMC differentiation, respectively. HES cells can also be used as an in vitro research model to examine vasculo- and angiogenesis mechanisms. Although the complex occurrences leading to angiogenesis are not completely understood yet, considerable interest is presently centered on the inhibition of new vascular growth to treat the spread of cancer. Therefore, implementing the method suggested herein, inhibition of spontaneous sprouting and network formation from human vascular cells was accomplished by blocking VE-cad (Fig. 3A and B). As a whole, this system facilitates the production of vascular lineage from hESCs and provides a platform for the study of human blood vessel development (Fig. 3C and D).
The data accumulated so far concerning the use of hESCs strengthen the claim that different markers seem to play an important role in human but not in mouse vasculogenesis (Kaufman et al., 2001; Levenberg et al., 2002). Therefore, it is noteworthy that previous works using murine systems are not necessarily predictive of human systems, rendering hESCs a significant tool for the study of human vascular development.
HIGHLIGHTS TO THE FUTURE
Apart from their invaluable contribution to the research on embryo development, hESCs play a major role in the exploration/investigation of transplantation medicine. As cardiomyocytes cannot be regenerated in adults, current therapeutic modalities for the treatment of end-stage heart failure are limited. The ability of hESCs to regenerate or repair damaged or ischemic cardiac tissue may solve this problem, but a few obstacles still hinder the clinical development of hESC-derived cardiovascular replacement therapy:
1Selection and expansion of pure vascular and heart cell populations
2Immune tolerance for allogenic cells
Selection of a pure cell population for transplantation can be resolved by genetic manipulation for the production and propagation of either pure populations of cardiovascular cells or cells that express suicidal genes permitting the ablation of the cells if they subsequently misbehave (Fareed and Moolten, 2002). As long-term maintenance of immunosuppressive therapy would limit successful clinical application, the creation of immune tolerance would enable the use of stem cell-derived therapy. Currently, hESC banks that represent the majority of tissue types reprogram the cells to carry the nuclear genome of the patient (Lanza et al., 2002) and generate hematopoietic chimerism (Bradley et al., 2002). There are only a few of the approaches that are being examined.
Upscaling platform technologies are under development. As the proliferative potential of ES-derived cardiomyocytes in vivo is probably limited, upscaling technologies would need to be applied to both undifferentiated and differentiated hESCs. These multiple hurdles will have to be overcome before transplantation of cardiovascular cells derived from hESCs becomes a clinical reality.