Pure Cardiomyocytes Research Articles

High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications.

Genetic enrichment of cardiomyocytes derived from mouse embryonic stem cells

Pluripotent embryonic stem cells (ESC) have the ability to differentiate into a variety of cell lineages in vitro , including cardiomyocytes. Successful applications of ESC-derived cardiomyocytes in cell therapy and tissue engineering were limited by difficulties in selecting the desired cells from the heterogeneous cell population. We describe a simple method to generate relatively pure cardiomyocytes from mouse ESCs. A construct comprising mouse cardiac α-myosin heavy chain (MHC) promoter driving the neomycin resistance gene and SV40 promoter driving the hygromycin resistant gene designated pMHCneo/ SV40-hygro, was stably transfected into mouse ESCs. The transgenic ESC line, designated MN6 retained the undifferentiated state and the potential of cardiogenic differentiation. After G418 selection, more than 99% of cells expressed α-sarcomeric actin. Immunocytological and ultrastructural analysis demonstrated that, the selected cardiomyocytes were highly differentiated. Our results represent a simple genetic manipulation used to product essentially pure cardiomyocytes from differentiating ESCs. It may facilitate the development of cell therapy in heart diseases. Key words: Embryonic stem cells, α-myosin heavy chain promoter, cardiomyocytes, differentiation, genetic enrichment.

293 MASS PRODUCTION OF Nkx2.5-POSITIVE CARDIAC PROGENITOR CELLS DERIVED FROM MOUSE EMBRYONIC STEM CELLS IN SLOW-TURNING LATERAL VESSEL FOR CELL TRANSPLANTATION AND DRUG TESTING

Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we developed an animal model for a single-step scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled slow-turning lateral vessel (STLV, Synthecon, Inc., Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. To enhance the yield of cardiac progenitor cells, mouse ES cells (HM1; 129Sv/Ola, Magin et al. 1992 Nucl. Acids Res. 20, 3795–3796) were targeted with the cardiac-specific mouse Nkx2.5 promoter driven enhanced fluorescent green protein (EGFP). Among 15 targeted colonies, which were characterised based on morphology, the ability to form EB, EGFP expression, and in vitro differentiation ability toward cardiomyocytes, 3 lines were further evaluated for the efficiency of cardiomyocyte production. The 3 lines were cultured in STLV bioreactor and compared with classical hanging drop (HD) and static suspension culture methods. Embryonic bodies at day 3 to 8 were collected and analysed by using fluorescence-activated cell sorting for markers of pluripotency (e.g. Oct-4, SSEA1, Nanog) and cardiac (e.g. Nkx2.5, Troponin T) lineage commitments. Data was analysed by one-way ANOVA and t-tests. The results showed that both level and kinetics of Nkx2.5 expression was dependent on culture conditions. The STLV and static suspension culture methods produced higher rates of Nkx2.5-positive cells on day 5 than that of HD (70 and 54 v. 30%, respectively). The STLV method produced a highly uniform population of efficiently differentiating EB in large quantities and resulted in the highest, 108 yield of cardiomyocytes in a single 110-mL STLV on day 4. In conclusion, the STLV method provides a technological platform for controlled large-scale generation of ES-cell-derived cardiomyocytes for clinical and industrial applications. In vivo transplantation tests of cardiomyocytes produced via STLV are currently underway. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205 and NKTH/KPI (NKFP_07_1-ES2HEART-HU OM-00202-2007), CHE-TRF senior scholarship, No. RTA 5080010 (M.T.), and the Thailand Commission on Higher Education [CHE-PhD-SW-2005-100 (S.R.), CHE-PhD-SW-RG-2007 (N.K.)].

Resveratrol reduces myofibroblast arrhythmogenicity

Background: Among grape skin polyphenols, trans-resveratrol (RES) has been reported to slow the development of cardiac fibrosis and to affect myofibroblast (MFB) differentiation. Because MFBs induce slow conduction and ectopic activity following heterocellular gap junctional coupling to cardiomyocytes, we investigated whether RES and its main metabolites affect arrhythmogenic cardiomyocyte-MFB interactions. Methods: Experiments were performed with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage-sensitive dyes. Long-term video recordings served to characterize drug-related effects on ectopic activity. Data are given as means ± S.D. (n = 4–20). Results: Exposure of pure cardiomyocyte strands to RES at concentrations up to 10 µmol/L had no significant effects on impulse conduction velocity (θ) and maximal action potential upstroke velocities (dV/dtmax). By contrast, in MFB-coated strands exhibiting slow conduction, RES enhanced θ with an EC50 of ~10 nmol/L from 226 ± 38 to 344 ± 24 mm/s and dV/dtmax from 48 ± 7 to 69 ± 2%APA/ms, i.e., to values of pure cardiomyocyte strands (347 ± 33 mm/s; 75 ± 4%APA/ms). Moreover, RES led to a reduction of ectopic activity over the course of several hours in heterocellular preparations. RES is metabolized quickly in the body; therefore, we tested the main known metabolites for functional effects and found them similarly effective in normalizing conduction with EC50s of ~10 nmol/L (3-OH-RES), ~20 nmol/L (RES-3-O-β-glucuronide) and ~10 nmol/L (RES-sulfate), respectively. At these concentrations, neither RES nor its metabolites had any effects on MFB morphology and α-smooth muscle actin expression. This suggests that the antiarrhythmic effects observed were based on mechanisms different from a change in MFB phenotype. Conclusions: The results demonstrate that RES counteracts MFB-dependent arrhythmogenic slow conduction and ectopic activity at physiologically relevant concentrations. Because RES is rapidly metabolized following intestinal absorption, the finding of equal antiarrhythmic effectiveness of the main RES metabolites warrants their inclusion in future studies of potentially beneficial effects of these substances on the heart.

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

BackgroundIt has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Methodology/Principal FindingsHere we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit+.Conclusions/SignificanceContradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency.

BioSpotlight

BioSpotlight

Global Expression Profile of Highly Enriched Cardiomyocytes Derived from Human Embryonic Stem Cells

Human embryonic stem cells (hESC), with their ability to differentiate into cardiomyocytes in culture, hold great potential for cell replacement therapies and provide an in vitro model of human heart development. A genomewide characterization of the molecular phenotype of hESC-derived cardiomyocytes is important for their envisioned applications. We have employed a lineage selection strategy to generate a pure population of cardiomyocytes (>99%) from transgenic hESC lines. Global gene expression profiling showed that these cardiomyocytes are distinct from pluripotent and differentiated hESC cultures. Pure cardiomyocytes displayed similarities with heart tissue, but in many aspects presented an individual transcriptome pattern. A subset of 1,311 cardiac-enriched transcripts was identified, which were significantly overpresented (p < .01) in the Gene Ontology (GO) categories of heart function and heart development. Focused analysis of the GO categories ion transport, sarcomere, and heart development uncovered a unique molecular signature of hESC cardiomyocytes. Pathway analysis revealed an extensive cardiac transcription factor network and novel peroxisome proliferator-activated receptor signaling components within the cardiac-enriched genes. Notably, approximately 80% of these genes were previously uncharacterized. We have evaluated the biological relevance of four candidates-Rbm24, Tcea3, Fhod3, and C15orf52-by in situ hybridization during early mouse development and report that all were prominently expressed in cardiac structures. Our results provide the fundamental basis for a comprehensive understanding of gene expression patterns of hESC cardiomyocytes and will greatly help define biological processes and signaling pathways involved in hESC cardiomyogenic differentiation and in human heart development.

Wnt3a-Induced Mesoderm Formation and Cardiomyogenesis in Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) into pure human cardiomyocytes (hESCMs) would present a powerful tool to further the creation of cell models designed to advance preclinical drug development. Here, we report a novel differentiation method to substantially increase hESCM yield. Upon early and transient treatment of hESCs with Wnt3a, embryoid body and mesendoderm formation is enhanced, leading to greater differentiation toward cardiomyocytes. Moreover, the generated beating clusters are highly enriched with cardiomyocytes (50%) and express genes characteristic of cardiac cells, providing evidence that these hESCMs are competent to develop in vitro into functional and physiologically relevant cardiomyocytes. In summary, this protocol not only has the potential to guarantee a renewable supply of enriched cardiomyocyte populations for developing novel and more predictive cell models, but it also should provide valuable insights into pathways critical for cardiac regeneration.

Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

BackgroundDevelopmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Methodology/Principal FindingsHere we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and α-myosin heavy chain (αMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.Conclusion/SignificanceThe protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

Cardiomyocyte Production in Mass Suspension Culture: Embryonic Stem Cells as a Source for Great Amounts of Functional Cardiomyocytes

Exploiting embryonic stem cell (ESC)-derived cardiomyocytes as a vital source for cell therapies and tissue engineering will depend on robust, large-scale production processes. Recently, we have reported stirring-controlled formation of embryoid bodies, enabling the generation of pure cardiomyocytes in 2-L scale. Expansion and differentiation of genetically engineered mouse ESCs was followed by antibiotic-based cardiomyocyte enrichment. Here we have investigated modification of various parameters aiming at process optimization in a 250-mL spinner flask system. Duration of the differentiation phase, timing of retinoic acid addition, and a slower medium exchange rate were found to be crucial to enhancing cardiomyocyte yield. Improved process conditions were consequently transferred to a 2-L controlled bioreactor. Employing a manual fill-and-draw medium change resulted in the formation of 0.86 x 10(9) cardiomyocytes in a single 2-L batch, thereby reproducing our previous findings. In contrast, an automated perfusion-based strategy enabled the production of 4.6 x 10(9) cardiomyocytes in a single run. This is significantly higher than previously reported and highlights the enormous process optimization potential in the scalable generation of ESC-derived cell lineages.

Preclinical assessment of cardiac toxicity

The exact prediction of the clinical behavior of drugs represents one of the most difficult duties in preclinical drug development. The use of cell-based assay systems underpins the development of many drug candidates, but owing to the artificial character of many of these systems, cell response and physiological behavior seem to be mutually exclusive. Embryonic stem cell-derived cells represent a system that may address the disconnect between the behavior of cultured cells and cells in situ. While undifferentiated ES cells allow standardization, expansion and genetic manipulation, the differentiated cells provide a reflection of the normal physiological image of their primary counterpart. We compare common models to detect cardiac toxicity with an assay system comprising in vitro differentiated pure cardiomyocytes.

Microprojections regulate proliferation and activity of cardiomyocytes derived from mouse embryonic stem cells

The goal was to test if the physical properties of the microenvironment control proliferation and activity of the mouse embryonic stem cell (mESC) progeny. Microtopographic features were fabricated by photolithography to create 15μm high projections spaced 80 or 500μm apart tetragonally in poly‐dimethyl‐silicone (PDMS) membranes. mESC differentiation began in hanging drops, followed by suspension culture, before the resulting embryoid bodies were disassociated and plated on either flat or the microtextured surfaces. The number of heterogeneous SC derivatives observed with phase microscopy was 60 ± 20% (n=3) on the 80μm microprojections compared to flat PDMS. Similar results were seen for pure cardiomyocytes derived using a puromycin resistant cassette incorporated into the NCX1 promoter. Only 43±12% (n=3) and 75±16% (n=5) of the cardiomyocytes were found on the 80μm and 500μm spaced microprojections, respectively, compared to the flat PDMS. The beating rate per minute of the cardiomyocytes was recorded by video microscopy and was 1.8±0.4 fold higher on the microprojections compared to the flat with surprising changes in coefficients of variance of 0.50 and 0.21 respectively (n=5). Results suggest that microtopography affects both expansion and beating characteristics of mESC progeny. T32HL007692 and HL 62426.

Highly enriched cardiomyocytes from human embryonic stem cells

Current efforts to direct differentiation of human embryonic stem cells (hESC) into a particular cell lineage usually lead to a heterogeneous cell population with only a fraction of the desired cell type present. We show the generation of an essentially pure population of human cardiomyocytes from hESC using lineage selection. A construct comprising the murine alpha-myosin heavy chain (alpha-MHC) promoter driving the neomycin-resistance gene was introduced into hES3 cells to generate stable transgenic lines. Transgenic hESC lines were differentiated into cardiomyocytes and subjected to G418 selection. Both G418-selected and non-selected cardiomyocytes were characterized by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The teratoma-forming potential of differentiated cells was assessed by injection of about 2 million cells into the hind limb muscle of SCID mice. Results After cardiac differentiation and antibiotic selection in a suspension culture process, more than 99% of the transgenic cells showed immunoreactivity to alpha-MHC and alpha-actinin; this enrichment efficiency was observed for independent transgenic cell lines. Quantitative RT-PCR analysis revealed high levels of enrichment for cardiac-specific messages in the selected population. Importantly, injection of selected cells into six SCID mice resulted in no apparent teratoma formation, in contrast to differentiated but non-selected controls. Our results represent a significant step toward scalable production of pure human cardiomyocytes from stable, expandable hESC lines that will facilitate the development of cell therapies, safety pharmacology and drug discovery.

Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes

To investigate whether hypoxia reoxygenation induces premature senescence in neonatal Sprague-Dawley (SD) rat cardiomyocytes. Cardiomyocytes were isolated from neonatal SD rat heart and identified by immunohistochemistry. The control cultures were incubated at 37 degree centigrade in a humidified atmosphere of 5% CO(2) and 95% air. The hypoxic cultures were incubated in a modular incubator chamber filled with 1% O(2), 5% CO(2), and balance N2 for 6 h. The reoxygenated cultures were subjected to 1% O(2) and 5% CO(2) for 6 h, then 21% oxygen for 4, 8, 12, 24, and 48 h, respectively. Cell proliferation was determined using bromodeoxyuridine labeling. The ultrastructure of cardiomyocytes was observed by using an electron microscope. beta-Galactosidase activity was determined by using a senescence beta-galactosidase Staining Kit. p16( INK4a ) and telomerase reverse transcriptase (TERT) mRNA levels were measured by real time quantitative PCR. TERT protein expression was determined by immunohistochemistry. Telomerase activities were assayed by using the Telo TAGGG Telomerase PCR ELISAplus kit. The initial cultures consisted of pure cardiomyocytes identified by immunohistochemistry. The proportion of BrdU positive cells was reduced significantly in the hypoxia reoxygenation-treated group (P< 0.01). Under the condition of hypoxia reoxygenation, mitochondrial dehydration appeared; p16( INK4a ) and TERT mRNA levels, beta-galactosidase activity, TERT protein expression and telomerase activities were all significantly increased (P< 0.01 or P< 0.05). These data indicate that premature senescence could be induced in neonatal SD rat cardiomyocytes exposed to hypoxia reoxygenation. Although TERT significantly increased, it could not block senescence.

Laser microdissection and capture of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived hyperoxia induces p21 in peri-infarct myocytes

Myocardial infarction caused by ischemia-reperfusion in the coronary vasculature is a focal event characterized by an infarct-core, bordering peri-infarct zone and remote noninfarct zone. Recently, we have reported the first technique, based on laser microdissection pressure catapulting (LMPC), enabling the dissection of infarction-induced biological responses in multicellular regions of the heart. Molecular mechanisms in play at the peri-infarct zone are central to myocardial healing. At the infarct site, myocytes are more sensitive to insult than robust fibroblasts. Understanding of cell-specific responses in the said zones is therefore critical. In this work, we describe the first technique to collect the myocardial tissue with a single-cell resolution. The infarcted myocardium was identified by using a truncated hematoxylin-eosin stain. Cell elements from the infarct, peri-infarct, and noninfarct zones were collected in a chaotropic RNA lysis solution with micron-level surgical precision. Isolated RNA was analyzed for quality by employing microfluidics technology and reverse transcribed to generate cDNA. Purity of the collected specimen was established by real-time PCR analyses of cell-specific genes. Previously, we have reported that the oxygen-sensitive induction of p21/Cip1/Waf1/Sdi1 in cardiac fibroblasts in the peri-infarct zone plays a vital role in myocardial remodeling. Using the novel LMPC technique developed herein, we confirmed that finding and report for the first time that the induction of p21 in the peri-infarct zone is not limited to fibroblasts but is also evident in myocytes. This work presents the first account of an analytical technique that applies the LMPC technology to study myocardial remodeling with a cell-type specific resolution.

Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control

It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 x 10(6) cells/mL after 9 days of differentiation. Approximately 30%-40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (alphaMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 x 10(9) cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications.

Adult ventricular cardiomyocytes: isolation and culture.

Isolated cardiomyocytes are a prerequisite to study the biology of cardiomyocytes. Efficient isolation is difficult, as these cells adhere firmly together in the heart and do not divide. Therefore, any experiment is restricted to the amount of calcium-tolerant, rod-shaped cardiomyocytes that can be initially isolated from the heart. This chapter gives detailed instructions on how ventricular cardiomyocytes can be isolated from an intact adult heart. The method is based on the principle of calcium-free perfusion with collagenase supplementation to disrupt cell-cell contacts in the heart, isolation and purification of cardiomyocytes from other cell types, and, finally, re-establishing a physiological cellular calcium concentration. The chapter also summarizes some commonly used adaptations to isolate cardiomyocytes from species different from rat.

Design of Artificial Myocardial Microtissues

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

Generation of confluent cardiomyocyte monolayers derived from embryonic stem cells in suspension: a cell source for new therapies and screening strategies

Cellular cardiomyoplasty is evolving as a new strategy to treat cardiac diseases. A prerequisite is a reliable source of pure cardiomyocytes, which could also help in the exploitation of recent advances in genomics and drug screening. Our goal was to establish a robust lab-scale process for the generation of embryonic stem (ES)-cell-derived cardiomyocytes in suspension. A 71 ES cell clone carrying a construct consisting of the alpha-cardiac myosin heavy chain (alphaMHC) promoter driving the neomycin resistance gene was used for antibiotic-driven cardiomyocyte enrichment. Rotating suspension culture was established to initiate embryoid body (EB) formation. To track growth and differentiation kinetics, cell count and flow cytometry for SSEA-I, E-cadherin (stem-cell marker)and sarcomeric myosin (cardiomyocytes marker) was performed. Oct4 expression was measured via real time (RT)-PCR. Cultures comprising 2.5-8 x 10(6) differentiating FS cells/mL were obtained after 9 days in rotating suspension. Upon G418 addition,vigorous contracting spheres, termed cardiac bodies (CB), developed. These cultures consisted of about 2.1 x 10(5) enriched cardiomyocytes/mL after 6- 10 days of selection. Suspensions comprising 90- 95%viable single cells were generated using an improved dissociation method. Seeding of cardiomyocytes with 7 x 10(4) cell/cm(2) resulted in a homogeneous monolayer of synchronously contracting cells. Myocyte specific immunohistochemistry indicated purity of > 99%. We have established a reliable lab-scale protocol to generate cultures of highly enriched cardiomyocytes in suspension. This will facilitate development of larger-scale processes for stem-cell based cardiomyocyte supply. An improved method is provided to derive vital suspensions of cardiomyocytes, which could be utilized for transplantation as well as for drug screening purposes.