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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Frank Pillekampa,b, Michael Reppelb, Olga Rubenchykb, Kurt Pfannkucheb, Matthias Matzkiesb, Wilhelm Blochc, Narayanswami Sreerama, Konrad Brockmeiera, Jrgen Heschelerb作者单位：aDepartment of Pediatric Cardiology, University of Cologne, Cologne, Germany;bInstitute of Neurophysiology, University of Cologne, Cologne, Germany;cDepartment of Molecular and Cellular Sports Medicine, German Sport University, Cologne, Germany & [0 \% Y- L7 v. {( f
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      【摘要】
   Human embryonic stem cell (hESC)-derived cardiomyocytes have been suggested for cardiac cell replacement therapy. However, there are no data on loaded contractions developed by these cells and the regulation thereof. We developed a novel in vitro transplantation model in which beating cardiomyocytes derived from hESCs (line H1) were isolated and transplanted onto noncontractile, ischemically damaged ventricular slices of murine hearts. After 2¨C3 days, transplanted cells started to integrate mechanically into the existing matrix, resulting in spontaneous movements of the whole preparation. Preparations showed a length-dependent increase of active tension. In transplanted early beating hESC-derived cardiomyocytes, frequency modulation by field stimulation was limited to a small range around their spontaneous beating rate. Our data demonstrate that this novel in vitro transplantation model is well suited to assess the mechanical properties and functional integration of cells suggested for cardiac replacement strategies.
      【关键词】 Pluripotent stem cells Myocardial contraction Cell transplantation Myocardial infarction Transplants6 v# {6 \5 O+ B/ m% \2 g! [
   INTRODUCTION
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Cell therapy is increasingly proposed as a treatment of ischemic heart failure. The main goal of transplanting cells into the injured myocardium is to reconstitute its contractile function . In this study, this novel model was used to characterize the contractile and integrative properties of early human ESC (hESC)-derived cardiomyocytes into ischemically damaged myocardium.0 ?, n& T; k' G7 `
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MATERIALS AND METHODS! e0 I  d3 J6 x4 {! r% D7 J7 l
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Culture of hESCs+ q7 h& x6 K- m, y9 u9 I1 n9 P4 T! B, m

The hESC line H1 (passage numbers between 30 and 70; WiCell Research Institute, Madison, WI, http://www.wicell.org) was imported according to German law and after approval by the Robert Koch Institute (Berlin) (number AZ 1,710-79-1-4-2). H1 cells were differentiated to spontaneously beating cardiac clusters as described previously . Briefly, H1 colonies were cultivated on CF1 feeder cell layers for approximately 7 days before they were transferred as clusters of approximately 500¨C800 cells onto cell layers of a visceral endoderm-like cell line (END-2) pretreated with mitomycin C (Serva GmbH, Heidelberg, Germany, http://www.serva.de). hESCs were cultured in knockout Dulbecco's modified Eagle's medium supplemented with 20% serum replacer (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 1 mmol/l L-glutamine, 1% nonessential amino acids, 90 µM ß-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 4 ng/ml basic fibroblast growth factor. Then, hESCs and END-2 cells were cocultured for up to 2 weeks without serum or serum replacer and without basic fibroblast growth factor and scored for the presence of beating areas from 5 days onward.3 @9 @" B! l. G6 s/ S
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Preparation of Ventricular Slices and Oxygen and Glucose Deprivation

Generation of ventricular slices was performed similarly as described previously for late-stage embryonic hearts  with the exception that D-glucose was replaced by an equimolar concentration of 2-deoxyglucose (10 mmol/l) to inhibit glucose metabolism. Oxygen tension was reduced by constant bubbling with pure nitrogen to induce hypoxia. After 20 hours of OGD, slices were washed twice in PBS to remove 2-deoxyglucose), transferred to Iscove's medium supplemented with 20% fetal calf serum, and stored in an incubator under normal, normoxic conditions (37¡ãC; 5% CO2, 21% O2) until coculturing with beating clusters derived from hESCs.+ a$ G, I0 w( d3 l

ATP Assay

ATP was quantified by a luciferase-driven bioluminescence assay (ATP-Bioluminescence Assay Kit CLS II; Roche Diagnostics GmbH, Mannheim, Germany, http://www.roche.de/diagnostics/index.htm). Slices were put in sonification solution (ethanol 70% vol/vol, 2 mmol/l EDTA), immediately frozen in liquid nitrogen, and stored at ¨C80¡ãC until further processing. Frozen slices were homogenized by sonification and mashed on ice before transfer into PBS to dilute ethanol and EDTA that might disturb the luciferase reaction. Finally, luciferase reagent was added and luminescence was measured (Sirius Luminometer V2.2, Berthold Detection Systems, Pforzheim, Germany, http://www.berthold-ds.com). Protein content was used as a reference and was determined with a protein assay based on the method of Bradford (BioRad Protein Assay; Bio-Rad, Hercules, CA, http://www.bio-rad.com).

Coculturing5 R4 q) C) U9 o
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Beating clusters of hESC-derived cardiomyocytes were excised using the tip of an injection cannula. Before excision, a typical beating cluster covered an area of 0.21 ¡À 0.02 mm2 of the culture well. Three to 10 beating clusters and the neonatal ventricular slice after OGD were then transferred into a custom-made well. This coculture well contained a small funnel-shaped cavity at its bottom to prevent beating areas from being washed away from the ventricular slice and was filled with 4 ml of Iscove's medium supplemented with 20% fetal calf serum, penicillin, and streptomycin. During the first 2¨C3 days, we avoided any movement of the culture dish; thereafter, culture medium was changed every 2nd¨C3rd day until experimentation.

Force Measurements+ G- ?# W2 _" Z/ C& T
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After coculturing for 4¨C10 days, the preparations consisting of the irreversibly ischemically damaged, noncontractile myocardial tissue and the hESC-derived beating areas were mounted on an isometric force transducer (Scientific Instruments GmbH, Gilching bei M¨¹nchen, Germany, http://www.si-gmbh.de). Slicing the hearts orthogonal to the long axis resulted in a tissue ring; the cavity of the left ventricle provided a preformed hole that eased mounting of the preparations onto the tips of two adjacent steel needles, a set-up very similar to the one used for force measurements of vascular rings. Length was increased stepwise to the length of maximal force development (Lmax). Contractions were recorded from spontaneously beating as well as from electrically stimulated preparations. Preparations were field-stimulated by silver electrodes (0.5¨C10 Hz, 5¨C15 V, stimulus pulse duration 5 ms) connected to a custom-made stimulator. The preparation was maintained at 37¡ãC and immersed in a dish filled with Iscove's medium without serum (Ca2  1.5 mmol/l). Electrical stimuli and analog signals from the force transducer (KG7A; range 0¨C5 mN, resolution 0.2 µN, resonance frequency 250¨C300 Hz) were amplified with a bridge amplifier (BAM7C; Scientific Instruments GmbH), and analog signals were transferred to an analog to digital board and recorded as well as analyzed using the software DasyLab Version 7.0 (National Instruments Corporation, Austin, TX, http://www.ni.com).

Tissue Processing, Immunohistological Staining, and Electron Microscopy  f: ]4 ~! Z3 G( ]: k

For immunohistological staining, preparations were fixed for 20 minutes in 99.8% methanol at ¨C20¡ãC or 120 minutes in 4% paraformaldehyde, washed with PBS, and incubated in 18% sucrose for 1 hour prior to embedding in Tissue Tek OCT (Sakura Finetek Japan Co., Ltd., Tokyo, http://www.sakura-finetek.com/top_e.html). After cryoslicing (7 µm), sections were placed on silanized slides. The primary antibodies were anti-pan-cadherin (1:500, C1821; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), anti-desmoplakin 1 and 2 (undiluted; Progen GmbH, Heidelberg, Germany, http://www.progen.de), anti-vimentin (1:200, V6630; Sigma-Aldrich), anti-cardiac -actinin (1:800, EA53; Sigma-Aldrich), and anti-human mitochondria (1:50, MAB1273; Chemicon International, Temecula, CA, http://www.chemicon.com). The specificity of the latter to differentiate between murine and human cells was shown with HEK293 cells and murine embryonic fibroblasts (Fig. 1).

Figure 1. Antibody specificity. Immunostaining with anti-human mitochondria antibody (purple) to demonstrate the specificity of the antibody for human, as opposed to murine, cells. Nuclei counterstained with Hoechst 33342. (A): Human embryonic epithelial kidney cells (HEK293) with an exposure time of 460 ms. (B): Mouse embryonic fibroblasts after an exposure time of 6,000 ms. Scale bars = 20 µm.

Goat anti-mouse, biotin-conjugated (1:400; Dako Denmark A/S, Glostrup, Denmark, http://www.dako.dk) and Alexa Fluor 647 and 546 (Invitrogen) were used as secondary antibodies. Nuclear staining was done with Hoechst 33342 (Sigma-Aldrich). Images were taken with an Axiovert 200; the image processing software was Axiovision Release 4.3 (both Carl Zeiss, Jena, Germany, http://www.zeiss.com). For electron microscopy, tissue was fixed in 4% paraformaldehyde for 120 minutes at room temperature. Afterwards, preparations were postfixed with 2% osmium tetroxide in 0.1 M PBS for 2 hours at 4¡ãC. Before embedding in araldite (Novartis International, Basel, Switzerland, http://www.novartis.com), the tissue was dehydrated in a graded series of ethanol. Ultrathin sections (60 nm) were mounted on formvar-coated copper grids, stained with 0.2% uranyl acetate and lead citrate, and then examined with a Zeiss EM 902 A electron microscope.

Statistical Analysis! R0 Z( m9 t& A# f! ]& {3 y

All data are expressed as means ¡À SEM unless otherwise stated. One-way analysis of variance was performed for each group by using Bonferroni's post hoc test. p values
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RESULTS

Induction of Severe Ischemic Injury and In Vitro Transplantation$ s% A+ X9 W3 L! m* f+ |
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After the slicing procedure (Fig. 2A, 2B), the majority of slices contracted spontaneously. After transfer of the slices to the hypoxia chamber (Fig. 2C) and 20 hours of OGD, hematoxylin and eosin staining (Fig. 2D), as well as electron microscopy (Fig. 2E), showed a severe disruption of structural integrity. OGD resulted in a complete loss of ATP (Table 1) and a complete loss of spontaneous beating (Table 2) of the murine tissue slices. To mimic cardiac cell therapy in vitro, we transplanted clusters containing early ESC-derived cardiomyocytes onto ventricular slices after these had been subjected to OGD. Within the first 2¨C3 days in the coculture well (Fig. 3A), beating clusters adhered loosely to the surface of the slices while still maintaining their original shape, resulting in a mushroom-like surface. During the following 4¨C7 days, this irregular surface smoothened and flattened and hESC-derived clusters filled fissures and cavities on the surface of the ventricular slice (Fig. 3B). At this time, even rigorous washing procedures could not detach the hESC-derived clusters any more. Contractions of the beating areas resulted in movements of the whole preparation, indicating a mechanical integration of the hESC-derived cardiomyocytes (supplemental online Video). This observation was corroborated by electron microscopy showing direct structural interaction between ESC-derived cells and the murine matrix, which might be responsible for force transmission (Fig. 3C, 3D). Ultrastructurally, we observed typical desmosomes and fasciae adherentes between hESC-derived cardiomyocytes (data not shown) and ESC-derived tissue stained positive for the desmosomal protein desmoplakin and pan-cadherin as a marker for adhesion junctions (Fig. 3E, 3F). In contrast, the morphological substrate of the mechanical interaction between the hESC-derived tissue and the damaged murine ventricular slice differed, given that no desmosomes or fasciae adherentes could be observed. Frequently, the extracellular matrix was visible in between the vital hESC-derived cells and the damaged myocardial tissue. Frequently, ESC-derived noncardiomyocytes formed an intermediate layer between the damaged tissue slice and the ESC-derived cardiomyocytes.5 K3 S# m+ R- A, t% a8 q- h  z

Figure 2. Oxygen and glucose deprivation (OGD) to simulate ischemia. Neonatal ventricular slice preparation and effect of OGD. (A): Neonatal murine heart (postnatal day 3). The gray plane illustrates the typical section plane. (B): Top view on a typical neonatal ventricular slice. (C): Drawing of the hypoxia chamber filled with glucose-deprived solution, bubbled with N2, and put into a water bath to guarantee a temperature of 37¡ãC. Hematoxylin and eosin staining (D) as well as an ultrastructural investigation (E) show a severe disruption of tissue integrity in slices after 20 hours of OGD. Scale bars = 100 µm (A, B), 20 mm (C), 20 µm (D), 5 µm (E).

Table 1. ATP concentrations of murine ventricular tissue slices without OGD as compared with slices subjected to 20 hours of OGD
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Table 2. Spontaneous contraction of murine ventricular tissue slices without OGD as compared with slices subjected to 20 hours of OGD! h0 C. e* P' z, r8 d2 g4 [

Figure 3. Morphology of the coculture of spontaneously beating human embryonic stem cell (hESC)-derived cardiomyocytes 10 days after transplantation onto an ischemically damaged ventricular slice. (A): Funneled culture well that prevents cocultures from being washed apart. (B): hESC-derived cell cluster (hESC) forming a smooth hump on the outer surface (O) of the remaining murine matrix (mMX) (see supplemental online video for beating behavior). (C): Ultrastructure of the outer surface (O) of the preparation that is coated by an hESC-derived cell (NCL, nucleus), showing a direct structural connection (D) to the murine matrix. (E, F): Immunostaining for (E) pan-cadherin and (F) desmoplakin in the hESC adjacent to the ischemically damaged murine matrix. (G¨CK): Preparation after 2 weeks of coculturing stained with anti--actinin to visualize areas with contractile proteins (yellow), anti-human mitochondria antibodies to verify the human phenotype of the grafted cells (green), Hoechst 33342 to counterstain the nuclei (blue), autofluorescence (Cy3 filter) to visualize the slice (gray), and anti-vimentin (red). Staining for -actinin and vimentin (G, I, J) and human mitochondria and vimentin (H, K) in directly following sections. (G, H): hESC migrating into the murine slice. (I): Infiltration of vimentin- and -actinin-positive cells along a fissure into the depth of the remnants of the murine slice. (J): Vimentin-rich, -actinin-negative cells covering small -actinin-positive myocytes with an early striated pattern. (K): Typical vimentin-rich cell deep within the damaged murine tissue. Scale bars = 200 µm (B), 2 µm (C), 0.4 µm (D), 100 µm (E¨CH), 20 µm (I), 10 µm (J), 5 µm (K).  ^$ o: N% F( M0 `

Immunohistochemical staining with an antibody highly specific for human mitochondria (Fig. 1) showed that the majority of hESC-derived cells used already existing fissures to infiltrate into the damaged murine tissue (Fig. 3G¨C3I). The transplanted cells showed various morphologies, including early human cardiomyocytes characterized by a high content of -actinin with an early striated pattern as well as numerous mitochondria (Fig. 3H, 3K). The whole slice was coated with a layer two to three cells thick of hESC-derived cells with variable vimentin staining without differentiation into cardiomyocytes (Fig. 3G, 3I, 3J). In the depth of the murine matrix, we detected predominantly vimentin-rich, -actinin-negative hESC-derived cells (Fig. 3H, 3K). Staining with the cell proliferation marker Ki-67 indicated that beating clusters contained proliferative cells even after several days of coculture.3 v, t. O) i% ^1 [

Isometric Force Measurements+ _1 w: _' \  c: Q% Z4 Z
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Isometric force measurements showed spontaneous contractions of the annular coculture preparations (Fig. 4A). We observed a dominant spontaneous beating frequency of 1.1 ¡À 0.1 Hz (range, 0.6¨C1.7 Hz) in preparations with clusters that started beating less than 10 days before coculturing. The developed force amounted to 4.9 ¡À 0.9 µN (n = 14). Twitch parameters (n = 7) are shown in Table 3. Slices cocultured with isolated fibroblasts did not develop any measurable force (n = 4). The parameters were obtained at spontaneous beating frequencies of 1.0¨C2.2 Hz. There was a nonsignificant correlation (p = .056) for preparations with a higher spontaneous beating rate to have a faster maximal relaxation velocity (normalized to the amplitude). When the length of the preparation was increased stepwise the stretch-induced enhancement of passive tension was accompanied by an increase of the active tension until the length of maximal force development was reached (Fig. 4B, 4C). Typically, the passive component was one to two magnitudes higher than the actively developed force. A further increase of the length of the preparation resulted in a mild decrease of the developed force paralleled by an overexpansion of the preparation. Slackening resulted in a reduction of active and passive force disproportionate to the reduction of the length. A subsequent second increase of the length could restore the previously observed passive and active force, albeit at an increased length.
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Figure 4. Length-tension relationship. Human embryonic stem cell (hESC)-derived cardiomyocytes transplanted onto irreversibly damaged ventricular tissue slices. (A): Ring-shaped ventricular slice mounted on two curved steel needles and stretched for isometric force measurements. Scale bar = 1 mm. (B): Representative diagram of the length-tension relationship during spontaneous beating. Black line, left y-axis: Stepwise increase of the tension with increasing length, predominantly due to an increase of passive tension with a small superimposed active component. Gray line, right y-axis: After subtraction of the passive component by high-pass filtering, the stepwise increase of the developed force becomes evident. (C): Length-tension relationship of the experiment shown above with open circles symbolizing active and black squares passive tension. The spontaneous beating frequency of this preparation remained stable throughout the whole experiment.
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Table 3. Force parameters after transplantation of clusters of human embryonic stem cell-derived areas onto ventricular slices that had been subjected to 20 hours of oxygen and glucose deprivation (n = 7)

To analyze the effect of temperature on the developed force, we changed the temperature slowly (* a. c9 ?  ?, j2 M4 W
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Figure 5. Temperature effects. (A, B): Temperature (upper panel), beating rate (middle panel), and developed force (lower panel). (A): Temperature changes are accompanied by changes of the spontaneous beating frequency but not of the developed force. (B): Control experiment with constant temperature. Corresponding superimposed isometric twitches. (C): Mild hypothermia is associated with a prolonged twitch duration, predominantly due to increased relaxation times. (D): Control.

Modulation of the frequency of the preparations by overpacing was limited to a small range of frequencies around the spontaneous beating frequency (Fig. 6A¨C6C). Spontaneous activity prevented lower frequencies, and higher stimulation rates remained unanswered or resulted in a 1:2, 1:3, or 1:4 pacing if the stimulation frequency was close to multiples of the spontaneous beating activity. Even an increase of the amplitude of the stimulation pulses to nonphysiologically high levels (up to 50 V, 10 ms) could not change this limited modulation of the frequency. In one preparation generated with older (beating for approximately 4 weeks), presumably higher differentiated clusters, higher stimulation rates (up to 4 Hz) were accepted./ G) A2 p8 Y: D" B

Figure 6. Force-frequency relationship. (A): Original traces of a typical preparation with a spontaneous beating rate of approximately 1.6 Hz. Only field stimulation close to the spontaneous beating rate resulted in triggered contractions, whereas stimulation with 1 or 2 Hz did not affect contractions. (B): Beating frequency (filled circles) and force amplitudes (open circles) of the same preparation. Triggered contractions (1:1) were observed only in a very small range of frequencies (gray background) that are similar to those during spontaneous activity. (C): Typical behavior during stimulation with a continuously increasing frequency in a different preparation. Again, triggered contractions can be observed only within a small range of frequencies (rectangle).

DISCUSSION
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We could demonstrate that beating clusters derived from hESCs integrate mechanically into the matrix of damaged myocardial tissue. In addition, these cells conferred force of contraction, as shown by isometric force measurements. This indicates the existence of a relevant interaction of transplanted cells with host matrix. We are convinced that this new tissue culture model is more relevant to the clinical situation than measurements of single cells  indicate that embryonic tissue has unique properties influencing survival and differentiation. Therefore, the use of embryonic cardiac tissue for coculturing might have been advantageous as compared with tissue slices generated from adult hearts or other organs. Experiments comparing different developmental stages as well as studies with tissue slices generated from mice with genetic modifications of extracellular matrix proteins should be helpful in elucidating the molecular mechanisms of the integration of stem cell-derived cells.% f2 {' q9 U# R9 D& i' _" w

Our model is based on OGD as a well-established model to mimic myocardial infarction in vitro . The model is simple to use and, because it is not restricted to a specific cell type or matrix, should be particularly useful for studies comparing the numerous cell types proposed for cardiac cell therapy. In addition, it should deepen our understanding of cell integration processes. These studies will be necessary for successful clinical applications of cell replacement strategies.  j( k  D7 Y4 U% M: v7 n

Similarly to cell-cell contacts within normal myocardium, junctions between hESC-derived cardiomyocytes have been described to be close and to exhibit intercalated discs with desmosomes and fasciae adherentes .9 D9 M* r) c; U  h$ t$ Y
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However, the structural interactions between hESC-derived cells, cardiomyocytes, or other cell types seem to be unspecific adhesion contacts. Frequently, extracellular matrix was inserted between the damaged tissue slice and the hESC-derived tissue. We assume that these contacts are mediated via integrins, because this is the most well-recognized class of extracellular matrix receptors .
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In our model, it took approximately 1 week until hESC-derived cardiomyocytes were fully integrated into the damaged myocardial tissue. In clear contrast to murine cells after transplantation in vivo .9 w; A+ v( K" Q; k
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Quantitatively, the developed force was very small. The number of cells was quite low as compared with the number of cardiomyocytes in physiological myocardial tissue. This is due to the fact that during early developmental stages, myofibrillar arrays are still irregular and only gradually mature into parallel arrays of myofibrils before ultimately aligning to densely packed sarcomeres that should augment force development . We could not prove the existence of a true descending limb of the length-tension relationship, because further lengthening of the preparations resulted in only a mild decrease of the active force but a severe overexpansion of the preparation. Given that active force development seemed to be hardly affected, lengthening of the preparation was most likely due to a plastic deformation of the ischemically damaged tissue slice and did not result from a disruption of the contact between slice and beating clusters.
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Twitch parameters were similar to those described for adult human ventricular muscle preparations .' x  M6 Q6 \% j; U( q
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Mild hypothermia resulted in prolonged contraction and relaxation times. Although in human nonfailing ventricular myocardium an increase of the force of contraction has been reported with hypothermia . In contrast to the experiments in adult human myocardium that were performed during electrical stimulation, our changes were accompanied by a severe drop of the spontaneous beating frequency, which probably is explained by the well-known temperature dependency of ion channels.2 f) H. L8 m" l+ ]. f+ v) v/ ]8 J$ Y, r* `
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CONCLUSION$ H  l6 k! S  O' l7 j! ~1 I
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hESC-derived cardiac clusters integrated in vitro into a natural cardiac matrix with all the characteristics of irreversible ischemic injury in vivo. The hESC-derived cardiomyocytes conferred force to the damaged myocardium and showed a positive length-tension relationship. Force developed by cardiac clusters transplanted early after onset of beating exhibited an immature phenotype. Future studies will have to aim at augmenting the developed force but also will have to improve integration and differentiation.# z4 Q: j3 h% n% c6 n
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DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS

We thank Christine Mummery and Robert Passier (Huprecht Laboratory, Utrecht, Netherlands) for kindly providing END-2 cells, Cornelia Böttinger for supplying us with hESC-derived cardiomyocytes, Moritz Haustein and Annette Köster for their skillful technical assistance, and Professor Gabriele Pfitzer for very helpful scientific discussions. We also thank Suzanne Wood for the secretarial assistance and the electronic and mechanic workshop of the institute for their technical support. This work was supported by the Koeln Fortune Program/Faculty of Medicine, University of Cologne (Grant 157/2003) (F.P.). F.P. and M.R. contributed equally to the manuscript.
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