Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue.

A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

Characterisation of electrophysiological conduction in cardiomyocyte co-cultures using co-occurrence analysis.

Cardiac arrhythmias are disturbances of the electrical conduction pattern in the heart with severe clinical implications. The damage of existing cells or the transplantation of foreign cells may disturb functional conduction pathways and may increase the risk of arrhythmias. Although these conduction disturbances are easily accessible with the human eye, there is no algorithmic method to extract quantitative features that quickly portray the conduction pattern. Here, we show that co-occurrence analysis, a well-established method for feature recognition in texture analysis, provides insightful quantitative information about the uniformity and the homogeneity of an excitation wave. As a first proof-of-principle, we illustrate the potential of co-occurrence analysis by means of conduction patterns of cardiomyocyte-fibroblast co-cultures, generated both in vitro and in silico. To characterise signal propagation in vitro, we perform a conduction analysis of co-cultured murine HL-1 cardiomyocytes and murine 3T3 fibroblasts using microelectrode arrays. To characterise signal propagation in silico, we establish a conduction analysis of co-cultured electrically active, conductive cardiomyocytes and non-conductive fibroblasts using the finite element method. Our results demonstrate that co-occurrence analysis is a powerful tool to create purity-conduction relationships and to quickly quantify conduction patterns in terms of co-occurrence energy and contrast. We anticipate this first preliminary study to be a starting point for more sophisticated analyses of different co-culture systems. In particular, in view of stem cell therapies, we expect co-occurrence analysis to provide valuable quantitative insight into the integration of foreign cells into a functional host system.

High-frequency electrical stimulation of cardiac cells and application to artifact reduction.

A novel modality for the electrical stimulation of cardiac cells is described. The technique is based on HF stimulation-burst of HF (1-25 kHz) biphasic square waves-to depolarize the cells and trigger action potentials (APs). HF stimulation was demonstrated in HL-1 cardiomyocyte cultures using microelectrode arrays, and the underlying mechanisms were investigated using single-cell model simulations. Current thresholds for HF stimulation increased at higher frequencies or shorter burst durations, and were typically higher than thresholds for single biphasic pulses. Nonetheless, owing to the decreasing impedance of metal electrodes with increasing frequencies, HF bursts resulted in reduced electrode voltages (up to four fold). Such lowered potentials might be beneficial in reducing the probability of irreversible electrochemical reactions and tissue damage, especially for long-term stimulation. More significantly, stimulation at frequencies higher than the upper limit of the AP power spectrum allows effective artifact reduction by low-pass filtering. Shaping of the burst envelope provides further reduction of the remaining artifact. This ability to decouple extracellular stimulation and recording in the frequency domain allowed detection of APs during stimulation-something previously not achievable to the best of our knowledge.

Low strength static magnetic field inhibits the proliferation, migration, and adhesion of human vascular smooth muscle cells in a restenosis model through mediating integrins ß1-FAK, Ca2+ signaling pathway.

The proliferation, migration, and adhesion of vascular smooth muscle cells (VSMCs) and their interactions with extracellular matrix are key features of atherosclerosis and restenosis. Recently, there has been evidence that magnetic fields exert multiple effects on the biological performance of cells and may aid in the treatment of vascular disease. However, the effect of a static magnetic field (SMF) on human VSMCs still remains unknown. In this study, we aimed to determine the effects of low strength SMF on human VSMCs in an in vitro restenosis model. A SMF was established using neodymium-yttrium-iron permanent magnet. Human umbilical artery smooth muscle cells (hUASMCs) were isolated and seeded to a fibronectin-coated plate to form an in vitro restenosis model and then exposed to a vertically oriented field of 5 militesla (mT). MTT, transwell, and adhesion assays were used to demonstrate that the proliferation, migration, and adhesion potential of hUASMCs were significantly decreased after exposure to 5 mT SMF for 48 h compared with a non-treated group. Meanwhile, confocal microscopy analysis was used to demonstrate that integrin ß(1) clustering was inhibited by exposure to 5 mT SMF. Furthermore, the phosphorylation of focal adhesion kinase (FAK) was markedly inhibited, and the upregulated cytosolic free calcium had been reversed (p < 0.05). However, the biological effects of low strength SMF on hUASMCs could be blocked by the administration of GRGDSP-the blockade of integrins. In conclusion, a low strength SMF can influence the proliferation, migration, and adhesion of VSMCs by inhibiting the clustering of integrin ß1, decreasing cytosolic free calcium concentration, and inactivating FAK. With further validation, SMFs may aid in attenuating abnormal VSMCs biological performance and has potential to block atherogenesis and prevent restenosis.

A device for separated and reversible co-culture of cardiomyocytes.

A novel technique is introduced for patterning and controllably merging two cultures of adherent cells on a microelectrode array (MEA) by separation with a removable physical barrier. The device was first demonstrated by separating two cardiomyocyte populations, which upon merging synchronized electrical activity. Next, two applications of this co-culture device are presented that demonstrate its flexibility as well as outline different metrics to analyze co-cultures. In a differential assay, the device contained two distinct cell cultures of neonatal wild-type and beta-adrenergic receptor (beta-AR) knockout cardiomyocytes and simultaneously exposed them with the beta-AR agonist isoproterenol. The beat rate and action potential amplitude from each cell type displayed different characteristic responses in both unmerged and merged states. This technique can be used to study the role of beta-receptor signaling and how the corresponding cellular response can be modulated by neighboring cells. In the second application, action potential propagation between modeled host and graft cell cultures was shown through the analysis of conduction velocity across the MEA. A co-culture of murine cardiomyocytes (host) and murine skeletal myoblasts (graft) demonstrated functional integration at the boundary, as shown by the progression of synchronous electrical activity propagating from the host into the graft cell populations. However, conduction velocity significantly decreased as the depolarization waves reached the graft region due to a mismatch of inherent cell properties that influence conduction.

Conduction analysis in mixed cardiomyocytes-fibroblasts cultures using microelectrode arrays.

Models for cardiac arrhythmia currently exist primarily in in-vivo and computer simulation form. Towards the development of such a model in-vitro, a better understanding of electrical conduction in heterogeneous cultures is required. Increasing ratios of cardiomyocytes and fibroblasts were cultured on 500x500 microm arrays of 36 microelectrodes to study the emergence and properties of action potential propagation in mixed cultures. A minimum ratio of 70% cardiomyocytes to 30% fibroblasts was found to be necessary for detection of electrical activity. However, the establishment of a continuous, homogeneous depolarization wave across the culture required a higher proportion of cardiomyocytes; even a 90:10 ratio was unable to consistently produce a unidirectional, uniform depolarization wave as is seen in controls. This model underlines the importance and sensitivity of tissue homogeneity in supporting electrical conduction, and is especially relevant to studies of arrhythmia (reentry) and stem cell grafts.

Modeling conduction in host-graft interactions between stem cell grafts and cardiomyocytes.

Cell therapy has recently made great strides towards aiding heart failure. However, while transplanted cells may electromechanically integrate into host tissue, there may not be a uniform propagation of a depolarization wave between the heterogeneous tissue boundaries. A model using microelectrode array technology that maps the electrical interactions between host and graft tissues in co-culture is presented and sheds light on the effects of having a mismatch of conduction properties at the boundary. Skeletal myoblasts co-cultured with cardiomyocytes demonstrated that conduction velocity significantly decreases at the boundary despite electromechanical coupling. In an attempt to improve the uniformity of conduction with host cells, differentiating human embryonic stem cells (hESC) were used in co-culture. Over the course of four to seven days, synchronous electrical activity was observed at the hESC boundary, implying differentiation and integration. Activity did not extend far past the boundary, and conduction velocity was significantly greater than that of the host tissue, implying the need for other external measures to properly match the conduction properties between host and graft tissue.

Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells.

Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 microA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in beta-MHCwithout increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation.

Cardiac differentiation of embryonic stem cells with point-source electrical stimulation.

The use of pluripotent stem cells as a means to repair damaged heart tissue has recently emerged as a promising, yet controversial therapy. Despite the different approaches and the variety of cell types used, many of these procedures have been met with mixed success. The lack of understanding of the differentiation and integration process, notably with respect to electrical signaling, significantly hampers the development of these therapies. A system was thus developed allowing the use of point source electrical stimulation on embryonic stem (ES) cells to study the effect of physiologically-relevant electrical stimulus. When modulating the amplitude of the stimulus over various differentiation stages of embryonic stem cells, differences in the proportions of cardiomyocytes to embryonic stem cells were observed through quantitative PCR. The use of this technique might have larger applications in understanding molecular pathways towards the regeneration process.

Temporal resolution of stimulation threshold: a tool for electrophysiologic analysis.

Electrical stimulation of cardiac cultures with closed-loop control permits the determination of threshold in real time. The temporal response of stimulation threshold and underlying cell membrane excitability is valuable information for understanding the complex electrophysiologic processes within cardiac cells and can aid in understanding the mechanisms and effects of pharmaceuticals or other stimuli. This work presents the temporal response of stimulation threshold measured using HL-1 cardiac myocytes when exposed to changes in temperature and extracellular potassium concentration. These changes mimic systemic alteration of excitability and conditions that can result from ischemia in the heart. The results demonstrate the efficacy of stimulation threshold as a physiologic indicator and illustrate transient effects with both fast and slow time constants that can be resolved using a system that determines stimulation threshold in real time.

Interleukin-1alpha induction of tensile weakening associated with collagen degradation in bovine articular cartilage.

OBJECTIVE: To determine whether interleukin-1alpha (IL-1alpha) induces tensile weakening of articular cartilage that is concomitant with the loss of glycosaminoglycans (GAGs) or the subsequent degradation of the collagen network. METHODS: Explants of young adult bovine cartilage obtained from the superficial (including the articular surface), middle, and deep layers were cultured with or without IL-1alpha for 1 week or 3 weeks. Then, portions of the explants were analyzed for their tensile properties (ramp modulus, strength, and failure strain); other portions of explants and spent culture medium were analyzed for the amount of GAG and the amount of cleaved, denatured, and total collagen. RESULTS: The effect of IL-1alpha treatment on cartilage tensile properties and content was dependent on the duration of culture and the depth of the explant from the articular surface. The tensile strength and failure strain of IL-1alpha-treated samples from the superficial and middle layers were lower after 3 weeks of culture, but not after 1 week of culture. However, by 1 week of culture, IL-1alpha had already induced release of the majority of tissue GAGs into the medium, without detectable loss or degradation of collagen. In contrast, after 3 weeks of culture, IL-1alpha induced significant collagen degradation, as indicated by the amount of total, cleaved, or denatured collagen in the medium or in explants from the superficial and middle layers. CONCLUSION: IL-1alpha-induced degradation of cartilage results in tensile weakening that occurs subsequent to the depletion of GAG and concomitant with the degradation of the collagen network.