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1Cardiovascular Research Laboratory, Department of Physiology and Biophysics, 2Department of Anatomy, and 3Department of Obstetrics and Gynecology, Rambam Medical Center, The Bruce Rappaport Faculty of Medicine and the 1Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, 31096 Haifa, Israel
Submitted 8 January 2003 ; accepted in final form 14 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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cell-cycle withdrawal; DNA synthesis; embryoid bodies; sarcomeres
Embryonic stem (ES) cells are pluripotent cell lines that were initially derived from the inner cell mass of mouse blastocyst-stage embryos (9, 23) and recently also from human blastocysts (28, 33). These unique cell lines are characterized by their capacity for prolonged undifferentiated proliferation in culture while retaining the capability to differentiate into derivatives of all three germ layers. During in vitro differentiation, the mouse ES cells were demonstrated to develop into specialized somatic tissues, including cardiomyocytes, and were shown to recapitulate many of the processes of early in vivo cardiac development (4, 12). Recently, a similar reproducible cardiomyocyte differentiating system was also established in the human model (16, 35). Dispersed cells isolated from spontaneous beating areas generated within the differentiating embryoid bodies (EBs) were demonstrated to possess the ultrastructural, molecular, and functional properties of earlystage cardiomyocytes (16). More recently, we (15) have demonstrated that this differentiating system is not limited to the derivation of individual cardiomyocytes but rather a functional syncytium is generated with spontaneous pacemaker activity and action potential propagation.
In the present study, we attempted to further characterize the ultrastructural and morphological changes associated with the differentiation and maturation of the human ES cell-derived cardiomyocytes. We also aimed to establish this unique differentiating system as an in vitro model to study human cardiomyocyte cell-cycle regulation. Our results show a reproducible temporal pattern of early cardiomyocyte proliferation, cell-cycle withdrawal, and ultrastructural maturation in this model. The detailed characterization of the human ES cell-derived cardiomyocytes cells may also possess an important clinical value because these unique cells represent an attractive source for the newly emerging cell therapy strategies aiming to regenerate functional myocardium.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The human ES cell line used in the present study was the National Institutes of Health-approved human ES cell clone (H9.2). This line was obtained from Prof. Joseph Itskovitz-Eldor (Technion University, Haifa, Israel) and has been used in several previous studies (1, 15, 16, 33).
To induce differentiation, the ES cells were dispersed to small clamps (3–20 cells) with the use of collagenase IV (Life Technologies, 1 mg/ml). The cells were then transferred to plastic petri dishes, where they were cultured in suspension for 7–10 days. During this period, the cells aggregated to form EBs, which were then plated on 0.1% gelatin-coated culture dishes and observed microscopically for the appearance of spontaneous contractions.
Preparation of samples for light and transmission electron microscopy. Spontaneously contracting foci within the EBs were mechanically dissected and fixed in 3% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C for 24 h. After fixation, samples were rinsed with 7.5% sucrose in the 0.1 M sodium cacodylate buffer (pH 7.4), postfixed in 1% OsO4 in the same buffer for 1 h, dehydrated in graded ethanol, and embedded in Epon 812.
For histomorphometric analysis, semithin epoxy resin-embedded sections (0.5 µm) were generated and stained with 0.1% toluidine blue in 1% sodium tetraborate (pH 12). Morphometric analysis was subsequently carried out using a computerized image-analysis system composed of an Olympus CH40 microscope fitted with a charge-coupled device camera (model Wat-202D, Watec; Tokyo, Japan) and using the Analysis DocU3 software (Soft Imaging System; Munster, Germany). The boundary of each myocyte was traced, and the cells' longest diameter, width, length-to-width ratio and area were determined. Several sections from seven EBs were examined for each of the three developmental groups.
For transmission electron microscopy, thin (60–90 nm) sections were prepared with the use of a diamond knife on a ultramicrotome (Nova; Bromma, Sweden). Sections were mounted on copper grids, stained with saturated uranyl acetate and 1% lead citrate (Merck), and examined with the use of a transmission electron microscope (model 100 SX, JEOL; Peabody, MA) operating at 80 kV.
Immunohistochemistry and autoradiography. Incorporation of [3H]thymidine by the human ES cell-derived cardiomyocytes was used to assess DNA synthesis by these cells. [3H]thymidine (New Life Science; Boston, MA) was added to culture medium of beating EBs at a final concentration of 10 µCi/ml. After incubation of 17 h at 37°C, the contracting EBs were rinsed three times with DMEM medium. The beating areas within the EBs were mechanically dissected and fixed immediately in 10% neutral-buffered paraformaldehyde overnight. Preparations were then dehydrated in graded alcohols (70–100%), cleared in chloroform, and embedded in paraplast (Sherwood Medical).
The 5-µm sections were deparaffinized and treated with 3% H2O2 in methanol for 20 min. Sections were then stained with the Histostain-SP kit (Zymed) according to the manufacturer's instructions. The primary antibodies that were used included mouse anti-cardiac troponin I (anti-cTnI; Chemicon, Temecula, CA), anti-sarcomeric 
-actinin mAbs (Sigma), and anti-nebulin antibodies (Sigma) at dilutions of 1:5,000, 1:50, and 1:100, respectively. For autoradiography, after immunohistological processing, the sections were immersed at 42°C in an autoradiographic emulsion solution (LM-1, Amersham) for 4 min. The sections were then air dried and kept in light-tight boxes for 12 days. The emulsions were developed with the use of a Kodak D-170 developer (18°C, 7 min). The sections were then fixed in 25% sodium thiosulfate (Sigma) for 10 min and examined microscopically. Cardiomyocytes were identified in the sectioned EBs by the positive cytoplasmatic staining with sarcomeric -actinin. All cardiomyocyte nuclei were clearly identified and then scored (labeling index) for the percentage of nuclei that contained silver grains. The procedure was repeated for EBs at various developmental stages (multiple sections from seven different EBs sampled from at least three different experiments for each developmental group).
To further evaluate cardiomyocyte cell-cycle activity, we utilized an additional cell-cycle marker, Ki-67. Whole EBs were fixed in 4% paraformaldehyde and blocked with PBS containing 3% normal goat serum for 60 min. Immunostaining was then performed using mouse anti-cTnI antibody (Chemicon) at a dilution of 1:5,000 and rabbit polyclonal anti-human Ki-67 antibody (Santa Cruz) at a dilution of 1:100 overnight at 4°C. Preparations were then incubated with Cy-2-conjugated anti-rabbit IgG and Cy-3-conjugated anti-mouse IgG (both from Chemicon) at a dilution of 1:100 for 1 h at room temperature. Cell nuclei were counterstained with the DNA dye To-Pro-3 (Molecular Probes; Eugene, OR). Confocal microscopy was performed using a Nikon Eclipse E600 microscope and Bio-Rad Radiance 2000 scanning system. The cardiomyocyte labeling index was then determined as the percentage of cardiomyocyte nuclei that were positively stained for Ki-67.
Statistical analysis. Data are expressed as means ± SD. The EBs were divided into three groups: 1) early stage: 7–20 days postplating, 2) intermediate stage: 21–35 days postplating, and 3) late stage: 36–60 days postplating. Multiple sections from seven EBs from at lease three different experiments were analyzed in each group. Differences between the groups in the morphometric measurements and labeling indexes were tested with the use of a Kruskal-Wallis one-way analysis of variance on ranks test. If found to be statistically significant, then post hoc analysis was performed with the use of the unequal Tukey's test. P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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At 27 days postplating, ultrastructural maturation continued and was characterized by the development of a sarcomeric organization. This was manifested by fusion of the round clusters of the electron-dense material (Z bodies) to form a continuous line, the nascent Z band (Fig. 1D). Parallel Z bands were demonstrated to confine the myofibrils in the typical sarcomeric pattern. The Z bands also served as anchoring points for branching sarcomeres. Despite this increased level of organization, the number of sarcomeres within the cells was still relatively low.
At 40 days postplating, there was a gradual increase in the number of myofibrils and in their degree of spatial organization (Fig. 2, A and B). The cells become elongated as their cytoplasm filled with sarcomeres (Fig. 2A). There was a clear increase in the amount of the electron-dense material in the Z band (Fig. 2B). However, even at this stage, different degrees of organization patterns could exist simultaneously in neighboring cells in the same EB and even within the same cell (Fig. 2A).
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At 60 days of postplating, we could note an additional increase in the amount and degree of organization of the sarcomeres within the cytoplasm (Fig. 2C). This can also be viewed in both longitudinal and transverse sections (Fig. 2D). Mitochondria were seen packed in close association with the myofibrils (Fig. 2C). At this stage, discrete A and I bands in addition to the Z bands could also be noted within the sarcomeres (Fig. 2C) and, in a minority of the cells, also M bands. Interestingly, the system of transverse (T) tubules, considered to develop very late during cardiomyocyte development, was not seen in any of the cells examined.
Morphometric analysis. Immunohistochemical studies demonstrated that the human ES cell-derived cardiomyocytes were generally concentrated in a single area that occupied a relatively small portion of the EB (Fig. 3, A and B). Within these beating regions, however, cardiomyocytes dominated and consisted of 30–60% of all cells (Fig. 3, A–D).
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Morphological analysis of the ES cell-derived cardiomyocytes revealed a developmental maturation process. The early-stage cardiomyocytes were relatively small, round, or rod shaped and overwhelmingly mononucleated (Figs. 3C). With maturation, the overall appearance of the human ES-derived cardiomyocytes changed to strands of more elongated cardiomyocytes (Figs. 3D). This was also accompanied by an increase in the cytoplasm-to-nuclear ratio. Interestingly, in contrast to the murine ES model (18), almost all cells (>99%) remained mononucleated.
Morphometric analysis of the ES cell-derived cardiomyocytes was accomplished by measuring the longest length, width, length-to-width ratio, and area of individual cells. The general tendency (Fig. 4) was a significant increase in the length and area of the cardiomyocytes in late-stage EBs (n = 94) compared (P < 0.05) with cells from the early (n = 87) and intermediate (n = 102) stages. Because no significant changes were noted in the cells' width, this tendency was also coupled with a significant increase in the cells' length-to-width ratio in late-stage EBs (Fig. 4).
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Proliferation and DNA synthesis. Assessment of the incorporation of [3H]thymidine was used to evaluate the DNA synthesis ability of the ES cell-derived cardiomyocytes during in vitro development. To ensure that the labeling index measurements were performed in cardiomyocytes, we initially had to develop a reliable assay capable of distinguishing these cells from other ES-cell derivatives. Preliminary experiments indicated that this could be readily accomplished by initial mechanical microdissection of the spontaneously contracting foci within the EBs to achieve cardiomyocyte-enriched specimens. Cardiomyocytes could then be identified by the positive staining of sarcomeric -actinin and absence of Nebulin (a skeletal muscle marker) immunosignal.
Figure 5, A–D, displays typical examples of simultaneous autoradiographic and immunohistochemistry studies of undifferentiated stem cells (Fig. 5A) and of contracting EBs of different developmental stages (Figs. 5, B–D). Cardiomyocytes could be identified by the positive -actinin immunoreactive signal (red-orange areas in Figs. 5, B–D). Note the lack of staining at the initial stem cell phase (Fig. 5A) and the gradual increase in the intensity of the signal from early-stage (Fig. 5B) to intermediate- (Fig. 5C) and late-stage cardiomyocytes (Fig. 5D). Incorporation of [3H]thymidine was be identified by the presence of silver grains over the cells' nuclei (Fig. 5, A–D). Positive stained (silver-black) nuclei were present in the majority of cells at the undifferentiated stem cell phase (Fig. 5A) and their number gradually decreased from early-stage (Fig. 5B) to intermediate-stage (Fig. 5C) cardiomyocytes. DNA synthesis was almost completely absent (lack of [3H]thymidine uptake) in late-stage cardiomyocytes (Figs. 5D).
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The observed developmental pattern in the labeling index during in vitro cardiomyogenesis was observed in all EB studied (Fig. 6). This analysis revealed that undifferentiated stem cells as well as EBs in suspension before cardiomyocyte induction were actively synthesizing DNA (labeling indexes >60%). A similar high-labeling index was also noted in early-stage cardiomyocytes with 60 ± 10% of the cells showing [3H]thymidine uptake. The labeling index gradually declined in intermediate-stage (21–35 days postplating) contracting EBs (36 ± 7%) and was reduced to <1% in late-stage EBs (over 36 days postplating).
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To further evaluate cell-cycle activity during in vitro cardiomyocyte development we utilized an additional marker of cell-cycle activity, Ki-67. Expression of Ki-67 is a prerequisite for cells to transverse the cell cycle and undergo cell division. Figure 5, E and F, shows typical confocal images of EBs at various developmental stages costained with anti-cTnI antibodies (red) and anti-Ki-67 antibodies (green nuclear staining). Note the high percentage of positively stained nuclei for Ki-67 in early-stage cardiomyocytes (Fig. 5E) and the absence of any significant staining in late-stage cardiomyocytes (Fig. 5F). Hence, similar to the [3H]thymidine experiments, the labeling index for Ki-67 was 55 ± 23% in early-stage cardiomyocytes (20 days postplating) and was reduced to 10 ± 16% (P < 0.05) in intermediate-stage cardiomyocytes (30-day EBs) and to 2 ± 2% (P < 0.05) in late-stage EBs (38 days postplating).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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In this study, we examined the developmental changes associated with the in vitro differentiation and maturation of human ES cell-derived cardiomyocytes. Our results demonstrate a reproducible temporal pattern of cardiomyocyte proliferation, cell-cycle withdrawal, and ultrastructural and morphological maturation. In addition to the significance of these results in characterizing this promising cell source for future cell therapy procedures, the current report also presents a new in vitro surrogate system to study the mechanisms underlying cell-cycle regulation and ultrastructural development as well as possible interventions aiming to modify these processes.
Ultrastructural and morphological development. In our previous report (16), we have demonstrated that human ES cells can differentiate to generate contracting cells with structural and functional properties typical of early-stage cardiomyocytes. In the current study, we expanded these observations by examining the process of cardiomyocyte ultrastructural and morphometric maturation in this in vitro model during a period of up to 60 days in culture. Our results show that the human ES cell-derived cardiomyocytes follow a maturation process that is roughly similar to that reported in vivo (5, 7, 17, 20, 22) and in the in vitro murine ES model (11, 12, 34). This process was characterized by gross morphological changes in which the relatively small and round cells situated in round accumulations in early-stage EBs gradually developed to larger and more elongated cells that tended to accumulate in strands in late-stage EBs. These morphological changes were coupled with a similar ultrastructural maturation process characterized by a progressive increase in the amount and organization of the contractile material. Hence, the initial pattern of irregular myofibrillar distribution in early-stage myocytes gradually organized into parallel myofibril arrays in more developed cells and ultimately resulted in the generation of well-defined sarcomeres with recognizable A, I, and Z bands in late-stage myocytes. Yet it is interesting to note that despite this developmental pattern the human ES cell-derived cardiomyocytes still did not reach the level of maturity, typical of adult cardiomyocytes. This was manifested, for example, by the lack of developed T tubule system.
The inability of the human ES-derived cardiomyocytes to reach the same degree of ultrastructural maturity as noted in the vivo adult heart may be related to a number of factors. First, although human ES-derived cardiomyocytes were followed in this study for a relatively long period (up to 60 days), it might still not be long enough to achieve full ultrastructural maturity. To this end, it is interesting to note that human cardiomyocytes derived from fetal hearts as late as after midterm still did not achieve full ultrastructural maturity and that myofibrillar development continued throughout the entire fetal period (17). Other possible factors may be related to the in vitro culturing conditions of the model and the lack of in vivo signals for hypertrophic growth. In particular, the lack of hemodynamic workload typical of in vivo working myocytes may be of key importance because cardiomyocytes grown in vivo in the absence of this workload were shown to lack appropriate cell alignment and ultrastrctural development (3).
The mechanisms controlling the complex process of myofibril assembly and sarcomeric organization are still not fully understood (10). Several models have been used to study these processes, including different primary cell cultures and in situ embryonic models, mostly in chicks and rats. The development of the human ES differentiating system may prove valuable for these types of studies because of the relatively reproducible ultrastructural developmental process observed here, the prolonged culturing capability of the model, and the ability to modify the ES cells genetically. The latter property may be of significant value because the genetic modification of these important structural proteins would otherwise lead to embryonic lethality.
Cardiomyocyte proliferation and DNA synthesis. Whereas numerous animal studies examined cardiomyocyte DNA synthesis and cell cycle activity during embryonic, fetal, and neonatal development (2, 25, 31), there is a paucity of similar data regarding during early human cardiac development (14). In the current study, the DNA synthesis ability of human ES cell-derived cardiomyocytes and their capability to undergo reduplication and cell-cycle withdrawal were examined. Our results demonstrate a high level of [3H]thymidine uptake in early-stage cardiomyocytes which was gradually reduced in intermediate-stage EBs and was almost completely absent in late-stage EBs. Cessation of DNA synthesis in our model and cell cycle withdrawal was followed by a stage characterized by morphological and ultrastructural maturation. A similar pattern was also noted when cycling myocytes were identified by the expression of Ki-67 in the nuclei. Although the exact function of Ki-67 is not clear, it is expressed in all proliferating cells in late G1, S, and G2M phases of the cell cycle (21, 29). One advantage of this marker is that it is not involved in DNA repair.
The cell-cycle changes observed in the human ES cell-derived cardiomyocytes follow a roughly similar pattern to that found in the murine ES model (18) with a temporal difference related most probably to the interspecies differences. The highly reproducible results obtained in both ES models and also in several in vivo animal studies (31) raises the possibility that cardiomyocytes may be intrinsically programmed with respect to the number of cell divisions that can occur in between cardiogenic induction and cell-cycle withdrawal or by an "intrinsic timer" (6) that may control cell-cycle withdrawal.
In the mouse, there is a transition from hyperplastic to hypertropic growth shortly after birth. This transition is coupled with a marked increase in myofibrillar density and the formation of binucleated cardiomyocytes. Several studies (8, 32) documented during this period a gradual decrease in radiolabeled thymidine incorporation leading to the suggestion that binucleation results from genomic duplication and kariokinesis in the absence of cytokinesis. Our study provides some conflicting results with respect to these findings. First, we noted cessation of DNA synthesis in the human ES cell-derived cardiomyocytes after 
40–50 days from the initiation of differentiation (7–10 days in suspension plus >36 days postplating). Although this process was longer than that observed in the mouse ES model, it is significantly shorter than the length of the human gestational period. Second, the number of binucleated cardiomyocytes observed after cessation of DNA synthesis was very low (<1% in a similar range as in the undifferentiated ES cells).
Although these differences may be inherent to the in vitro nature of the model, there is some evidence to suggest similar in vivo phenomena. It is interesting to note that in a study that examined cardiomyocyte DNA synthesis during murine fetal and neonatal development, two temporally distinct phases of DNA synthesis were documented (32). The first phase occurred during early fetal life and was associated exclusively with cardiomyocyte proliferation and was followed by cessation of cardiomyocytes reduplication in late fetal life. During the neonatal period a second phase of DNA synthesis was observed, which was associated with the process of binucleation. The termination of DNA synthesis in our model may thus relate to the first phase in the above study. The paucity of binucleation in the human ES-derived cardiomyocytes may also relate to the first phase of DNA synthesis that was associated exclusively with reduplication in the murine study (32). It is also interesting to note that in general, the percentage of binucleated or multinucleated adult human cardiomyocytes (20%) (24) is much lower than that observed in the mouse heart (30) and that the number of binucleated cells in the mouse ES model was lower than that observed in vivo (18).
The reproducible results observed in this study also suggests that the human ES differentiating system may be utilized as a possible in vitro surrogate system to study the mechanisms involved in cardiomyocyte DNA synthesis, reduplication, and cell-cycle regulation as well as of molecular interventions aiming to modify these processes. The ability to intervene in these processes may be of considerable therapeutic value given the limited regenerative capacity of the adult heart. To this end, it is interesting to note that the mouse ES model has already been utilized to study the possible effects of such interventions, taking advantage of its ability to generate pure cardiomyocytes, its reproducible proliferative capacity, and the fact that it is readily amenable to cotransfection with multiple expression constructs (13, 26).
Another important implication of the current study relates to the possible utilization of the human ES cell-derived cardiomyocytes in future cell replacement strategies. The proliferative capabilities of the ES cell-derived cardiomyocytes may have a major impact on the ultimate success of this strategy because it may directly affect the number of cells available for transplantation as well as the size, survival, and possible integration of the in vivo cell graft (27). Combining the recently described human ES technology with gene transfer strategies aiming to modify cell-cycle properties may provide a particular attractive approach for improving the ultimate success of these newly emerging therapeutic paradigms.
Limitations of described model. Although the results of the current study suggest that the human ES cardiomyocyte differentiating system may serve as a unique in vitro model to study human cardiac tissue, it also holds some limitations. First, cardiomyocytes, although being highly concentrated in a single area, consist of only a minority of the cells within the entire EB. In addition, based on data from the mouse model, the ES cell-derived cardiomyocytes are expected to represent a mixed group of atrial- and ventricular-like cells (12). These limitations may be partially overcome in the future by the use of double labeling with well characterized antibodies or by establishing genetically modified ES clones expressing a selectable marker (antibiotic resistance gene, green fluorescent protein, etc.) under the control of a cardiac-specific promoter to generate pure cardiomyocyte cultures (19).
The second limitation of the model stems from its in vitro nature, which may be different from the in vivo settings, and may thus influence ultrastructural maturation and cell cycle activity. Some properties that may differ in the in vitro settings include the different availability of cell nutrients within the contracting EB, the presence of adjacent nonmyocyte proliferating cells, the lack of possible paracrine, humoral, neuronal, and other in vivo signals, and the lack of hemodynamic load.
Despite these limitations, the current study provides the first characterization of the developmental changes in the cell cycle activity and ultrastructural properties during the in vitro differentiation and maturation of human ES cell-derived cardiomyocytes. Our results demonstrate a reproducible temporal pattern of early cardiomyocyte proliferation, cell cycle withdrawal, and ultrastructural maturation in this model. The results of the current study may have important implications for future cell replacement and tissue engineering strategies using these unique cells and for the in vitro modeling of human cardiac tissue.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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