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Cardiovascular Research 2003 58(2):435-443; doi:10.1016/S0008-6363(02)00730-7
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Copyright © 2003, European Society of Cardiology

Survival and function of mouse embryonic stem cell-derived cardiomyocytes in ectopic transplants

Kohei Johkuraa,*, Li Cuib, Akihiro Suzukia, Ruifeng Tengb, Akiko Kamiyoshic, Shintaro Okamuraa, Suguru Kubotab, Xu Zhaob, Kazuhiko Asanumaa, Yasumitsu Okouchia, Naoko Ogiwaraa, Yoh-ichi Tagawac and Katsunori Sasakia,b

aDepartment of Anatomy and Organ Technology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan
bInstitute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan
cInstitute of Experimental Animals, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan

* Corresponding author. Tel.: +81-263-372590; fax: +81-263-373-093. kohei{at}sch.md.shinshu-u.ac.jp

Received 2 May 2002; accepted 21 October 2002


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: Embryonic stem cell-derived cardiomyocytes are a useful source for cell transplantation into the heart, as well as for tissue engineering of the extracardiac vascular system. The present study was designed to investigate the survival and contractile function of embryonic stem cell-derived cardiomyocytes around large blood vessels to assess the feasibility of their ectopic use for future engineering of cardiovascular tissues. Methods: The mouse embryonic stem cell-derived cardiomyocytes were transplanted into the retroperitoneum of the adult nude mice, and the myocardial tissues that developed were characterized by electrophysiological and histological techniques. Results: Macroscopic and electrophysiological analyses showed spontaneously contracting transplants in the host retroperitoneum 7 and 30 days after transplantation. Immunohistochemistry detected developing cardiomyocytes in the transplants on Day 7, which formed the myocardial tissues. They were positive for cardiac troponin I, cadherin, connexin 43, and proliferating cell nuclear antigen, but negative for {alpha}-smooth muscle actin. Vascular formation was discernible in the transplant tissues. By Day 30, more mature myocardial tissues had been established in the transplants. Electron microscopic study emphasized that the transplant tissues comprised cardiomyocytes, in which myofibrils with organized sarcomeres were observed. Desmosomes, fasciae adherens and gap junctions were evident in the cellular junctions. Conclusions: The cardiomyocytes derived from the mouse ES cells were demonstrated to be viable and function in the ectopic site of the host retroperitoneum up to Day 30, following a process of proliferation and differentiation. Vascularization and host perfusion beneficial for the survival of the cardiomyocytes occurred in the transplants.

KEYWORDS Contractile function; Developmental biology; Histo(patho)logy; Myocytes; Stem cells; Transplantation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Embryonic stem (ES) cells, derived from the inner cell mass of the blastocyst-stage early mammalian embryos [1,2], are expected to become a powerful tool for future regenerative medicine due to their capacity of self-renewal and pluripotency [3]. They can proliferate indefinitely and differentiate into derivatives of all three primary germ layers in vitro [4]. In terms of clinical benefit, this dual potency provides the source for cellular replacement or transplantation in a variety of organs [5,6].

Cardiomyocytes have been derived from various stem cells such as hematopoietic stem cells [7], bone marrow cells [8,9], embryonal carcinoma cells [10] and ES cells [2,11–15]. They have been provided for the in vitro study of the early stage of cardiomyogenesis [16,17], and also for intracardiac grafting to assess their therapeutical availability [18]. Meanwhile, fetal cardiomyocytes have been demonstrated to have the capacity to survive and increase in the cardiac tissue where they were engrafted [19–21]. This outcome means that transplantation of developing cardiomyocytes can regenerate damaged cardiac tissue. Likewise, when injected into the post-infarcted heart, the cardiomyocytes developed from the above stem cells were functionally integrated with the host myocardial tissues, indicating their ability to enhance myocardial repair and contribution to heart function [7–9,22–24].

The ectopic transplantation of developing cardiomyocytes into the connective tissue of adult animals has been tested, and established the feasibility of grafting cardiomyocytes into the scar tissue caused by the myocardial infarction [25]. We have emphasized an alternative application for ectopic transplantation of developing cardiomyocytes, namely, the creation of a vascular pump by surrounding the vasculature with myocardial tissue. In our recent study, cultured cardiomyocytes of mouse embryo origin were transplanted around the host abdominal aorta [26]. As a result, the myocytes were able to survive and contract in the site receiving the host perfusion. The aim of this transplantation was to obtain basic data on the structural and functional properties of the ectopic transplants derived from cultured cardiomyocytes. The bio-pump can be used as an aid in impaired heart function, and in the generation of a new circulation system independent of the host heart. An ectopic formation of the vascularized myocardial grafts for cardiomyoplasty is also considered a possibility on the basis of the same technique. These concepts should be supported in the future by tissue engineering coupled with the use of pluripotent stem cells as the source of the donor cells. To this end, verification of the viability and function of the ES cell-derived cardiomyocytes in the ectopic sites is prerequisite.

In the present study, generation of the ectopic transplants of the mouse ES cell-derived cardiomyocytes was attempted in the host retroperitoneum to evaluate their viability and contractility in the site around the abdominal aorta and inferior vena cava. The ES cell-derived cardiomyocytes in transplant tissues were characterized by electrophysiological and immunohistochemical approaches, as well as by ultrastructural technique. This study opens a new potential application for ES cell-derived cardiomyocytes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Animals and cells
The investigation conforms with Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). BALB/c nu/nu male mice on postnatal week 5 (n = 5 in each experimental group) were used as the host (Charles River Japan, Yokohama, Japan). Mouse ES cell line, E14.1, and a line developed from 129/sv strain (Cell and Molecular Technologies, Phillipsburg, NJ, USA) were used as the source of the donor cardiomyocytes. The E14.1 line was kindly donated by Dr Nobuaki Yoshida, The Institute of Medical Science, University of Tokyo.

2.2 Cell culture
ES cells were cultured in 5% CO2 at 37°C for 72 h in Dulbecco's modified minimal essential medium (GIBCO, Grand Island, NY, USA) supplemented with 20% FCS (HyClone, Logan, UT, USA), 100 µM non-essential amino acids (GIBCO), 100 µM sodium pyruvate (GIBCO), 100 µM 2-mercaptoethanol (Sigma, St. Louis, MO, USA), and 103 units/ml of leukemia inhibitory factor (LIF; Chemicon, Temecula, CA, USA) on feeder cells of mitomycin C-inactivated STO fibroblasts. After dissociation with 0.05% tripsin–EDTA, the cells were resuspended in Iscove's modified Dulbecco's medium (IMDM, GIBCO) containing 20% FCS, 100 µM non-essential amino acids, 1 mM sodium pyruvate, and 100 µM 2-mercaptoethanol without LIF in a concentration of 1000 cells/50 µl-drop, and cultured in 5% CO2 at 37°C for 5 days using the hanging drop method [13,27]. The embryoid bodies formed in the drops were replated onto the gelatin-coated 96-well microplate with one embryoid body/well, and cultured in the same medium for 3–7 days. The beating cardiomyocytes generated in the colony were collected with collagenase treatment, and used for cell transplantation. Some embryoid bodies were cultured on gelatin-coated glass coverslips for histological analyses.

2.3 Immunocytochemistry
After rinsing with phosphate-buffered saline at 37°C, the embryoid body outgrowths on the gelatin-coated glass coverslips were fixed in 4% paraformaldehyde/0.1 M phosphate buffer, pH 7.4 for 1 h at room temperature. They were then rinsed three times with 20 mM phosphate-buffered saline, pH 7.4. Prior to immuno-staining, the specimens were treated in a microwave at 100°C for 5 min in 10 mM citrate Na, pH 6.0 for antigen retrieval. The embryoid body outgrowths were doubly stained with specific antibodies for connexin 43 (rabbit polyclonal, Sigma) and cardiac troponin I [28] (mouse monoclonal, Chemicon). First, the former antibody was detected by goat anti-rabbit IgG conjugated with Alexa fluor 568, and subsequently, following blocking treatment with normal rabbit serum, the latter was detected by rabbit anti-mouse IgG conjugated with Alexa fluor 488. Fluorescent labeled antibodies were purchased from Molecular Probes (Eugene, OR, USA). Specimens were observed with an Olympus FLUOVIEW confocal laser scanning microscope (CLSM) equipped with Ar and He/Ne lasers.

2.4 Cell transplantation
The host mice were anesthetized with intraperitoneal injection of pentobarbital sodium solution (0.04 mg/gbw). The cardiomyocytes collected from approximately 100 embryoid body outgrowths were resuspended within IMDM with the same supplements as above at 4°C, and injected into the host retroperitoneum using a 1.0 ml syringe with a 26-gauge needle under a Leica M651 microsurgery microscope.

2.5 Electrophysiological analysis
The retroperitoneal transplants in the anesthetized host were exposed after survival times of 24 h (Day 1), 7 days (Day 7), and 30 days (Day 30). The electrocardiographs of the host heart and transplant were recorded simultaneously employing a multi-channel recorder PowerLab equipped with Chart v3.6 software (ADInstruments, Mountain View, CA, USA). The electrodes were located on the surface of beating areas in the transplants, and forefeet of the host.

2.6 Immunohistochemistry
The transplants from each time point were fixed in 4% paraformaldehyde/0.1 M phosphate buffer, pH 7.4 for 24 h at 4°C, and embedded in paraffin. Serial sections were cut at a thickness of 5 µm in each transplant. After deparaffinization and rehydration, the sections were stained with specific antibodies against cardiac troponin I, {alpha}-smooth muscle actin ({alpha}-SMA, mouse monoclonal, Progen Biotechnik, Heidelberg, Germany), pan-cadherin (mouse monoclonal, Sigma), connexin 43 and proliferating cell nuclear antigen (PCNA, mouse monoclonal, NeoMarkers, Union City, CA, USA) employing avidin–biotin–peroxidase complex method followed by diaminobenzidine reaction. The above antigen retrieval was performed prior to immunostaining except in the case of PCNA staining. The specimens were observed with a Nikon Microphot-FXA light microscope after counterstaining with methyl green solution. Control staining was performed omitting the step of staining with primary antibodies. Some specimens were stained for routine histology (H&E).

2.7 Electron microscopic study
Small pieces of the transplants were fixed in 2.5% glutaraldehyde/45 mM cacodylate HCl, pH 7.2 overnight. After rinsing three times in 180 mM sucrose/80 mM cacodylate HCl, pH 7.2 at 4°C for 3 h, the specimens were postfixed in 1% osmium tetroxide/0.1 M sodium cacodylate buffer, pH 7.2 for 90 min at 4°C, dehydrated in a graded series of ethanol and embedded in epoxy resin. Ultrathin sections of the specimens were stained with uranyl acetate and lead citrate, and observed with a JEOL JEM-1200 transmission electron microscope (TEM) at an accelerating voltage of 80 kV.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 ES cell-derived cardiomyocytes
A total of 3 to 7 days after expansion, the embryoid body outgrowths were observed to comprise multidifferentiated cells in which spontaneously beating cardiomyocytes were detected (Fig. 1a). Each population of the cardiomyocytes displayed synchronized beating. The cardiomyocytes positive for cardiac troponin I and connexin 43 were observed in the colonies by CLSM (Fig. 1b). Cardiac troponin I was detected on the myofibrils in the cytoplasm in a striated pattern, while connexin 43 was observed on the cell membranes exhibiting a punctate appearance.


Figure 1
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  		Fig. 1 Cardiomyocytes in the embryoid body outgrowths 5 days after expansion. (a) Hoffman modulation contrast image of the colony. The spontaneously beating areas of the cardiomyocyte are indicated with arrowheads. Bar=250 µm. (b) Immunofluorescence double staining of the ES cell-derived cardiomyocytes for cardiac troponin I (green) and connexin 43 (red). Cardiac troponin I is evident in the myofibrils in a striated pattern, while punctate staining for connexin 43 is detected in the cell–cell junctions (arrowheads). Bar=10 µm.

 
3.2 Macroscopic and electrophysiological analyses of the transplants
At the time of cell transplantation, cardiomyocyte suspension was successfully injected into the retroperitoneum adjacent to the large blood vessels (Fig. 2a). On Day 1, whitish transplants were observed in the host retroperitoneum (Fig. 2b). No discernible beating was detected in either the macroscopic or electrophysiological analyses. On Day 7, the transplants had grown with spontaneously beating areas (Fig. 2c). Vascular formation was observed in the transplants. The electrocardiograph showed that the transplants were contracting independently from the host heartbeat (Fig. 3a). The contraction was rather irregular and there appeared to be several foci in the transplants. On Day 30, large masses of transplants with beating areas were observed (Fig. 2d). The electrocardiograph showed that several foci of contraction existed, and that the contraction was more regular at each focus in comparison with that on Day 7 (Fig. 3b).


Figure 2
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  		Fig. 2 Macroscopic images of the transplants in the host retroperitoneum. (a) An image of the site just after receiving the injection of ES cell-derived cardiomyocytes. Injected suspension of cardiomyocytes (arrow) is observed in the retroperitoneum over the abdominal aorta adjacent to the inferior vena cava. (b) Day 1. A whitish transplant can be seen adjacent to the inferior vena cava (arrow). (c) Day 7. A transplant with spontaneously beating areas is observed (arrow). Vasculatures are discernible in the superficial region of the transplant. (d) Day 30. A mass of transplant has grown in the retroperitoneum (arrow). Beating areas are observed in the superficial region of the mass. IVC, inferior vena cava; A, abdominal aorta. Bar=5 mm.

 

Figure 3
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  		Fig. 3 Electrocardiogram of the transplant on Day 7 (a) and Day 30 (b). (a) Action potential of the spontaneous beating is detected on the surface of the transplant (top). Spikes with several different morphologies are detected in the electrogram. The contraction is rather irregular with an average rate of 66.7 beats/min (bpm), and is independent of the host heartbeat (bottom). (b) A regular contraction with an average rate of 171.4 bpm is detected in an electrogram of the transplant. Small spikes from different foci are still seen. Bar=500 ms.

 
3.3 Light microscopic observation of the transplants
On Day 1, the inside of the transplants was still filled with dissociated cells, but the margin adjacent to the host tissues exhibited organized tissues (Fig. 4a). The tissues contained cardiomyocytes positive for cardiac troponin I (Fig. 4b). By Day 7, the myocardial tissues had been formed in the transplants (Fig. 4c). The remainder appeared to be multidifferentiated tissues, in which cystic structures had formed. The cardiomyocytes were polygonal in shape, and positive for cardiac troponin I (Fig. 4e). They were simultaneously positive for connexin 43 and cadherin in their cell membranes, displaying a circumferential expression pattern (Fig. 4d, f). The cadherin, presumably N-isoform, and connexin 43 formed the components of the fascia adherens [29] and gap junction [16], respectively. The nuclei of cardiomyocytes displayed variable staining intensities for PCNA, exhibiting their proliferative activity in the transplants (Fig. 4g). Most of the cardiomyocytes were negative for {alpha}-SMA, a protein provisionally expressed during cardiomyogenesis (Fig. 4h). The smooth muscle tissues that were positive for {alpha}-SMA were associated with the vasculatures, including the blood capillaries in the myocardial tissues (Fig. 4h). These vasculatures exhibited well-differentiated structures with a thin endothelium and a relatively large luminal diameter. They contained erythrocytes, indicating the establishment of successful perfusion by the host. On Day 30, a large part of the transplants exhibited multidifferentiated tissues, but the myocardial tissues were observed near the surface of the transplants (Fig. 5). The cardiomyocytes acquired an elongated shape with extended myofibrils. They were positive for cardiac troponin I (Fig. 5b), connexin 43 (Fig. 5a) and cadherin (Fig. 5c), but completely negative for PCNA, indicating the end of proliferation (Fig. 5d).


Figure 4
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  		Fig. 4 Immunohistochemical staining of the transplant tissues on Day 1 (a, b) and Day 7 (c–h). (a) Injected cells have organized tissues (arrowheads) adjacent to the host inferior vena cava (IVC). A: abdominal aorta H&E. Bar=50 µm. (b) An aggregation of cardiomyocytes positive for cardiac troponin I can be seen in the transplant tissues (arrow). Bar=50 µm. (c) The transplant tissue is observed near the abdominal aorta (A), in which myocardial tissues positive for cardiac troponin I have been formed (arrows). Bar=0.5 mm. (d) Cell membranes of the cardiomyocytes are positive for connexin 43 in a circumferential pattern. Bar=50 µm. (e) Myofibrils positive for cardiac troponin I are irregularly arranged in the myocardial tissue. (f) Junctions of the cardiomyocytes are positive for cadherin. Most of the cardiomyocytes are not elongated. (f) Variable intensities of nuclear staining for PCNA are observed in the cardiomyocytes, exhibiting their proliferative activity. (h) The cardiomyocytes are almost negative for {alpha}-SMA. The smooth muscle cells positive for {alpha}-SMA are associated with the vasculatures. *Vasculatures in the myocardial tissues.

 

Figure 5
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  		Fig. 5 Immunohistochemical staining of the transplant tissues on Day 30. (a) Punctate appearance of positive staining for connexin 43 is observed in the junctions of the cardiomyocytes. (b) Regular arrangement of cardiac troponin I-positive myofibrils can be seen in the myocardial tissues. (c) Junctions of cardiomyocytes are positive for cadherin. (d) The cardiomyocytes exhibit negative staining for PCNA, indicating the termination of their proliferation. Bar=50 µm.

 
3.4 Electron microscopic observation of the transplants
The TEM observation further emphasized that the ES cell-derived transplant tissues comprised cardiomyocytes (Fig. 6). On Day 7, the cardiomyocytes had myofibrils with the definitive ultrastructures of an organized sarcomere, although they were thin and irregularly arranged in the cytoplasm (Fig. 6a). The cellular junctions, composed of fasciae adherens, desmosomes and gap junctions interconnected the neighboring cells (Fig. 6a, b). Actin filaments comprising the myofibrils were concentrated in the fasciae adherens. On Day 30, elongated cardiomyocytes with long and wide bundles of myofibrils were observed. The myofibrils with a definitive sarcomere were regularly arranged along the long axis of the myocytes (Fig. 6c). Cell–cell junctions were evident between the cardiomyocytes (Fig. 6d).


Figure 6
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  		Fig. 6 Ultrastructure of the cardiomyocytes in the transplant tissues on Day 7 (a, b) and Day 30 (c, d). (a) Myofibrils with organized sarcomeres are evident in the cytoplasm of the ES cell-derived cardiomyocyte. The sarcomere between Z-lines (Z) is obvious with the A and I bands. The fasciae adherens (arrowheads) and desmosome (arrow) can be seen in the cellular junction. Inset: Note both the longitudinal and cross images of the myofibrils (*) in the same cell. (b) The gap junction (arrow) as well as the fasciae adherens (arrowheads) are observed in the cellular junction of the cardiomyocytes. (c) Myofibrils with organized sarcomeres are more prominent in the cardiomyocytes than those on Day 7. They are regularly arranged in the cytoplasm. Z:Z-line. (d) Fasciae adherens can be seen in the junction of the cardiomyocytes (arrows). Bars=1 µm.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
The present study provided the electrophysiological and histological evidence to support that the ES cell-derived cardiomyocytes formed the myocardial tissues in the adult host retroperitoneum. These myocytes were demonstrated to be viable and contractile up to Day 30 in the ectopic site around the large blood vessels following their proliferation and differentiation. Some of their aspects on Day 7 were similar to those of immature cardiomyocytes, e.g. the round shape, circumferential expression of cadherin and connexin 43, random arrangement of myofibrils, and proliferative activity, whereas on Day 30, the cardiomyocytes exhibited a more differentiated property judging from their morphology and the results of immunostaining. The definitive ultrastructures of the organized sarcomeres confirmed the contractile function of the cardiomyocytes. The fasciae adherens, desmosomes and gap junctions demonstrated by immunohistochemical and ultrastructural studies established that these cardiomyocytes acquired the structural and electrical integration to form functioning myocardial tissues. It was also noteworthy that the vascular formation and the host perfusion essential for myocardial survival took place in the transplant tissues. The origin of the vasculatures, however, remains to be elucidated, i.e. whether angiogenesis by the host or vasculogenesis by the transplants contributed to the new formation of vasculatures. Given the pluripotency of the ES cells, one cannot exclude the possibility that precursor cells, such as angioblasts, are generated in the embryoid body outgrowths and contribute to the vascular formation in the transplants in a vasculogenic process. More work will be necessary to verify their origin using donor cells labeled with a genetic marker.

The ability of spontaneous contraction, the intrinsic and the most prominent feature of the cardiomyocytes, will be useful not only in myocardial repair [7–9,18,22,23], but in engineering a variety of tissues. We have proposed a new application of the cardiomyocytes transplantation, namely, the creation of an extracardiac pump by surrounding the vasculatures with myocardial tissues. In our recent study, cultured cardiomyocytes of mouse embryo origin transplanted around the host abdominal aorta were able to survive and contract in the site, and receive the host perfusion [26]. The results of the present study were consistent with our findings above, expanding the potency of the ectopic transplantation of the cardiomyocytes by means of ES cells as their source.

In the present study, a cardiomyocyte-rich preparation was enzymatically dissociated by collagenase from the embryoid body outgrowths, and provided for the transplantation. However, contamination of the donor cells with the residual ES cells or cells in alternate lineages was likely to occur, such as might cause the formation of multidifferentiated structures. The tumorigenic activity of the ES cells was recognized to be serious in the present study, i.e. even when the embryoid body outgrowths were transplanted after beating cardiomyocytes had appeared, teratomas were formed in the host retroperitoneum. These findings suggest that the in vitro differentiation of the embryoid body outgrowths, in which induction of the cardiomyocytes occurs, is not sufficient for transplantation unless the exclusive selection of the cardiomyocytes is established. An antibiotic selection by introducing a fusion gene comprising {alpha}-myosin heavy chain promoter and sequences encoding hygromycin resistance into the ES cells is now proceeding in our laboratories. In addition, a fusion gene of β-galactosidase and G418 resistance linked to cytomegalovirus promoter was situated in the downstream of the selection region as the graft marker described above. It is important to recognize that although further purification of the donor cells is necessary, the present study is the first demonstration of the viability and contractile function of ES cell-derived cardiomyocytes in an ectopic site other than the heart tissues, which will lead to developing new therapeutical approaches for cardiovascular diseases.

Time for primary review 25 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
We thank Mr Kiyokazu Kametani and Ms Kayo Suzuki, Research Center for Instrumental Analysis, Shinshu University, for their outstanding technical assistance. This work was supported by a grant from the Japan Foundation of Cardiovascular Research.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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