Correction: Rehnelt et al. Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation. Int. J. Mol. Sci. 2017, 18, 2634.

The authors wish to make the following corrections to this paper [...].

An impedance-based approach using human iPSC-derived cardiomyocytes significantly improves in vitro prediction of in vivo cardiotox liabilities.

Current in vitro approaches to cardiac safety testing typically focus on mechanistic ion channel testing to predict in vivo proarrhythmic potential. Outside of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, structural and functional cardiotoxicity related to chronic dosing effects are of great concern as these effects can impact compound attrition. Development and implementation of an in vitro cardiotoxicity screening platform that effectively identifies these liabilities early in the discovery process should reduce costly attrition and decrease preclinical development time. Impedence platforms have the potential to accurately identify structural and functional cardiotoxicity and have sufficient throughput to be included in a multi-parametric optimization approach. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) have demonstrated utility in cardiac safety and toxicity screening. The work described here leverages these advantages to assess the predictive value of data generated by two impedance platforms. The response of hIPSC-CMs to compounds with known or predicted cardiac functional or structural toxicity was determined. The compounds elicited cardiac activities and/or effects on "macro" impedance often associated with overt structural or cellular toxicity, detachment, or hypertrophy. These assays correctly predicted in vivo cardiotox findings for 81% of the compounds tested and did not identify false positives. In addition, internal or literature Cmax values from in vivo studies correlated within 4 fold of the in vitro observations. The work presented here demonstrates the predictive power of impedance platforms with hIPSC-CMs and provides a means toward accelerating lead candidate selection by assessing preclinical cardiac safety earlier in the drug discovery process.

Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation.

Side effects on cardiac ion channels causing lethal arrhythmias are one major reason for drug withdrawals from the market. Field potential (FP) recording from cardiomyocytes, is a well-suited tool to assess such cardiotoxic effects of drug candidates in preclinical drug development, but it is currently limited to the spontaneous beating of the cardiomyocytes and manual analysis. Herein, we present a novel optogenetic cardiotoxicity screening system suited for the parallel automated frequency-dependent analysis of drug effects on FP recorded from human-induced pluripotent stem cell-derived cardiomyocytes. For the expression of the light-sensitive cation channel Channelrhodopsin-2, we optimised protocols using virus transduction or transient mRNA transfection. Optical stimulation was performed with a new light-emitting diode lid for a 96-well FP recording system. This enabled reliable pacing at physiologically relevant heart rates and robust recording of FP. Thereby we detected rate-dependent effects of drugs on Na⁺, Ca2+ and K⁺ channel function indicated by FP prolongation, FP shortening and the slowing of the FP downstroke component, as well as generation of afterdepolarisations. Taken together, we present a scalable approach for preclinical frequency-dependent screening of drug effects on cardiac electrophysiology. Importantly, we show that the recording and analysis can be fully automated and the technology is readily available using commercial products.

Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium.

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)-derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6-10-fold because of induction of proliferation on purification. Long-term engraftment (4-5 months) was observed when co-transplanting selected ES cell-derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell-derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

Scalable selection of hepatocyte- and hepatocyte precursor-like cells from culture of differentiating transgenically modified murine embryonic stem cells.

Potential therapeutic applications of embryonic stem cell (ESC)-derived hepatocytes are limited by their relatively low output in differentiating ESC cultures, as well as by the danger of contamination with tumorigenic undifferentiated ESCs. To address these problems, we developed transgenic murine ESC clones possessing bicistronic expression vector that contains the alpha-fetoprotein gene promoter driving a cassette for the enhanced green "live" fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under established culture conditions these clones allowed for both monitoring of differentiation and for puromycin selection of hepatocyte-committed cells in a suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]). When plated on fibronectin, the selected eGFP-positive cells formed colonies, in which intensely proliferating hepatocyte precursor-like cells gave rise to morphologically differentiated cells expressing alpha-1-antitrypsin, alpha-fetoprotein, and albumin. A number of cells synthesized glycogen and in some of the cells cytokeratin 18 microfilaments were detected. Major hepatocyte marker genes were expressed in the culture, along with the gene and protein expression of stem/progenitor markers, suggesting the features of both hepatocyte precursors and more advanced differentiated cells. When cultured in suspension, the EB-derived puromycin-selected cells formed spheroids capable of outgrowing on an adhesive substrate, resembling the behavior of fetal mouse hepatic progenitor cells. The established system based on the highly efficient selection/purification procedure could be suitable for scalable generation of ESC-derived hepatocyte- and hepatocyte precursor-like cells and offers a potential in vitro source of cells for transplantation therapy of liver diseases, tissue engineering, and drug and toxicology screening.

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.

Synchronized electromechanical integration recording of cardiomyocytes.

Cardiac issues are always one of major health problems that attract wide attention by the public. It is urgent to explore a preclinical strategy to efficiently prevent the life-threatening arrhythmias by precisely assessing the cardiac excitation-contraction behavior. Conventional label-free asynchronous strategies are difficult to synchronously record and precisely match the excitation and contraction signals in vitro, while label-based strategies generally present pharmacological adverse effects and phototoxicity that significantly interfere the natural excitation and contraction signals. Both types of strategies preclude to exactly understand how cardiac excitation-contraction coupling changes in quantitative and coherent detail when dysfunctions occur. Here, we show a label-free synchronized electromechanical integration detection strategy that can synchronously monitor electrical and mechanical signals of cardiomyocytes over a long period of time by an integrated microelectrode-interdigitated electrode (ME-IDE). ME-IDE can detect subtle changes in electromechanical integration signals induced by drugs that target excitation-contraction coupling. Moreover, electromechanical integration delay is explored to specifically recognize the sodium channel inhibition. Furthermore, biomimetic electronic pacemaker function provides an alternative way to efficiently assess the drug-induced arrhythmia using refractory period of cardiomyocytes.

Elevated serum levels of CC thymus and activation-related chemokine (TARC) in primary Hodgkin's disease: potential for a prognostic factor.

The CC thymus and activation-related chemokine (TARC) is a protein, which is highly expressed by Reed-Sternberg cells in Hodgkin's disease and is found in the majority of Hodgkin's disease patients. Within several trials conducted by the German Hodgkin study group, 62 Hodgkin's disease patients were elected based on availability of serum samples post and prior therapy to assess TARC levels by ELISA. TARC levels from 33 patients with continuous complete response (CCR), 20 patients with relapse, and nine patients with progressive disease (PD) were correlated with freedom from treatment failure and survival. As defined in healthy donors (mean value +/- 2x SD), a TARC level of >500 pg/mL was considered as elevated. The median TARC levels of all patients at baseline and after completed primary treatment were 5,803 pg/mL (range, 116-73,074 pg/mL) and 663 pg/mL (50-24,709 pg/mL), respectively. TARC levels of patients with PD were higher than those of patients with CCR at baseline and after therapy. Baseline TARC correlated significantly with stage (P = 0.019), erythrocyte sedimentation rate (P = 0.004), leukocyte count (P < 0.001), and lymphocyte count (P = 0.026). A TARC level of >2,000 pg/mL after completed treatment was a significant risk factor for poorer survival (P = 0.02) but not for relapse. In conclusion, monitoring serum TARC levels in Hodgkin's disease patients may add valuable information about therapy success in Hodgkin's disease patients, especially those with PD and should therefore be prospectively evaluated in future trials.

Phase I/II combined chemoimmunotherapy with carcinoembryonic antigen-derived HLA-A2-restricted CAP-1 peptide and irinotecan, 5-fluorouracil, and leucovorin in patients with primary metastatic colorectal cancer.

PURPOSE: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen-derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells. EXPERIMENTAL DESIGN: HLA-A2-positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-gamma intracellular cytokine assays were done to evaluate CTL reactivity. RESULTS: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1-specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1-specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen-specific CD8+ cells decreased by an average 14%. CONCLUSIONS: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1-specific T cells were observed in 47% of patients after vaccination.

Cellular cardiomyoplasty improves survival after myocardial injury.

BACKGROUND: Cellular cardiomyoplasty is discussed as an alternative therapeutic approach to heart failure. To date, however, the functional characteristics of the transplanted cells, their contribution to heart function, and most importantly, the potential therapeutic benefit of this treatment remain unclear. METHODS AND RESULTS: Murine ventricular cardiomyocytes (E12.5-E15.5) labeled with enhanced green fluorescent protein (EGFP) were transplanted into the cryoinjured left ventricular walls of 2-month-old male mice. Ultrastructural analysis of the cryoinfarction showed a complete loss of cardiomyocytes within 2 days and fibrotic healing within 7 days after injury. Two weeks after operation, EGFP-positive cardiomyocytes were engrafted throughout the wall of the lesioned myocardium. Morphological studies showed differentiation and formation of intercellular contacts. Furthermore, electrophysiological experiments on isolated EGFP-positive cardiomyocytes showed time-dependent differentiation with postnatal ventricular action potentials and intact beta-adrenergic modulation. These findings were corroborated by Western blotting, in which accelerated differentiation of the transplanted cells was detected on the basis of a switch in troponin I isoforms. When contractility was tested in muscle strips and heart function was assessed by use of echocardiography, a significant improvement of force generation and heart function was seen. These findings were supported by a clear improvement of survival of mice in the cardiomyoplasty group when a large group of animals was analyzed (n=153). CONCLUSIONS: Transplanted embryonic cardiomyocytes engraft and display accelerated differentiation and intact cellular excitability. The present study demonstrates, as a proof of principle, that cellular cardiomyoplasty improves heart function and increases survival on myocardial injury.

Immunomagnetic enrichment and detection of micrometastases in colorectal cancer: correlation with established clinical parameters.

PURPOSE: Micrometastatic disease in bone marrow is of prognostic significance in colorectal cancer patients. However, detection rates of standard immunocytology are relatively low. We used magnetic activated cell sorting (MACS), a highly sensitive method, to increase detection rates and correlated the presence of cytokeratin (CK)-expressing cells with clinical parameters. PATIENTS AND METHODS: Bone marrow was obtained from 51 consecutive patients with newly diagnosed colorectal adenocarcinoma who underwent primary surgery and 18 control subjects. International Union Against Cancer (UICC) stage I disease was diagnosed in 11 patients, stage II disease was diagnosed in 14 patients, stage III disease was diagnosed in 12 patients, and stage IV disease was diagnosed in 14 patients. CK-positive cells were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to CK 7 and 8. RESULTS: CK-positive cells were found in 33 (65%) patients and were absent in 18 (35%). Four of 11 (36%) patients with UICC stage I disease, nine of 14 (64%) with stage II diease, eight of 12 (67%) with stage III disease, and 12 of 14 (86%) with stage IV disease were CK-positive. Epithelial cells were more frequently found in pT3/4 (72%) than in pT1/2 (36%) tumors (P =.026), but there was no difference for lymph node status. CK-positive patients had a higher chance for elevated carcinoembryonic antigen (85% v 15%, P = NS) and CA 19-9 levels (92% v 8%, P =.019). There were no significant differences in CA 72-4, sex, age, tumor grading, or tumor localization regarding the presence of CK-positive cells. All control subjects were CK-negative. CONCLUSION: In searching for micrometastases in colorectal cancer patients, we have achieved high detection rates by using MACS. The presence of these cells correlated significantly with tumor stage, tumor extension, and the tumor marker CA 19-9.

[Progressive multifocal leukoencephalopathy following azathioprine and steroid treatment of pulmonary sarcoid disease]. / Progressive multifokale Leukenzephalopathie. Schwerwiegende Komplikation einer Immunsuppression.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system following reactivation of the John-Cunningham-Virus (JVC) in an immunocompromised host. This rare condition is characterized by rapid progressing neurologic symptoms often leading to death. In the following, we report on a rapid evolving deterioration of mental status due to PML in an 53-year-old man during treatment of pulmonary sarcoid disease using azathioprine and steroids. In contrast to reported lethal outcomes, our patient experienced a slow recovery of his cognitive impairment and later on of his palsy following termination of immunosuppression.

Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies.

The detection of isolated tumor cells (ITC) in the bone marrow of patients with epithelial malignancies is an independant prognostic factor for several entities as breast cancer, colorectal cancer or non-small lung cancer. However, with conventional immunocytology using Ficoll density gradient and APAAP staining, only a small proportion of the bone marrow samples can be scanned for cytokeratin-positive (CK+) cells. To improve detection rates, we evaluated the enrichment of ITC by magnetic activated cell sorting (MACS) compared to regularly stained cytospins. Recovery experiments with a CK+ breast cancer cell line (SKBR3) were performed to calculate the MACS enrichment rate. Bone marrow was obtained by aspiration from 20 patients with carcinomas of epithelial origin and from 17 controls. ITC were enriched and stained with magnetically labeled CAM 5.2 antibodies directed to cytokeratin 7 and 8. MACS of SKBR3 seeded in peripheral blood revealed average recovery rates of 62% and 48% and average enrichment factors of 104-fold and 8139-fold of the CK+ cells after one and after two separations, respectively. After immunomagnetic enrichment, CK+ cells were detected in 16 of 20 (80%) cancer patients, whereas only 7 (35%) patients showed CK+ cells without magnetic enrichment (P = 0.002). Ten of twelve (83%) patients with metastatic disease (stage M1) and six of eight (75%) patients without any overt metastases (M0) had CK+ cells in their bone marrow. None of the negative controls showed any CK+ cells. Enrichment with magnetically labeled anti cytokeratin antibodies increases the detection rate of epithelial cells in bone marrow of cancer patients compared to immunocytology.

Influence of genomics on cancer vaccine development from guess to prediction.

After more than 100 years since the first adjuvant for a cancer vaccine was described and more than a decade since the first tumor antigen has been molecularly cloned, it seems possible that cancer vaccines might be integrated into the standard care of cancer patients. Exciting new technologies concerning tumor antigen discovery, vaccine delivery and formulation define the basis for enormous efforts in academia as well as in the pharmaceutical and biotech industry. With the unveiling of the human genome additional targets will emerge that could further enhance vaccine efficacy, specificity and clinical applicability. Most likely therefore, tumor antigen targets which are widely expressed in cancer will be of advantage over patient-oriented approaches due to their favorable cost-to-benefit ratio. Some widely expressed candidate tumor antigens and methods to discover additional widely expressed tumor antigens are discussed here. While the armamentarium of potential tools to cancer vaccine development seems to be endless, only those that are scientifically sound yet economically reasonable will in the end have a chance to become clinically useful cancer vaccines.

Minimized cell usage for stem cell-derived and primary cells on an automated patch clamp system.

INTRODUCTION: Chip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons. METHODS: We established application protocols for CHO cells, IPS cell-derived neurons (iCell® Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U®, Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5 µl cell suspension per well for densities between 55,000 cells/ml and 400,000 cells/ml depending on cell type resulted in good cell capture. RESULTS: We present a new cell application procedure optimized for the Patchliner achieving>80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application. DISCUSSION: Our new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability.

Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.

Dynamic monitoring of beating periodicity of stem cell-derived cardiomyocytes as a predictive tool for preclinical safety assessment.

BACKGROUND AND PURPOSE: Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process. EXPERIMENTAL APPROACH: Beating of cardiomyocytes was measured by a recently developed microelectronic-based system using impedance readouts. We used mouse stem cell-derived cardiomyocytes to obtain dose-response profiles for over 60 compounds, including ion channel modulators, chronotropic/ionotropic agents, hERG trafficking inhibitors and drugs known to induce Torsades de Pointes arrhythmias. KEY RESULTS: This system sensitively and quantitatively detected effects of modulators of cardiac function, including some compounds missed by electrophysiology. Pro-arrhythmic compounds produced characteristic profiles reflecting arrhythmia, which can be used for identification of other pro-arrhythmic compounds. The time series data can be used to identify compounds that induce arrhythmia by complex mechanisms such as inhibition of hERG channels trafficking. Furthermore, the time resolution allows for assessment of compounds that simultaneously affect both beating and viability of cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: Microelectronic monitoring of stem cell-derived cardiomyocyte beating provides a high throughput, quantitative and predictive assay system that can be used for assessment of cardiac liability earlier in the drug discovery process. The convergence of stem cell technology with microelectronic monitoring should facilitate cardiac safety assessment.

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Potential risks of bone marrow cell transplantation into infarcted hearts.

Cellular replacement therapy has emerged as a novel strategy for the treatment of heart failure. The aim of our study was to determine the fate of injected mesenchymal stem cells (MSCs) and whole bone marrow (BM) cells in the infarcted heart. MSCs were purified from BM of transgenic mice and characterized using flow cytometry and in vitro differentiation assays. Myocardial infarctions were generated in mice and different cell populations including transgenic MSCs, unfractionated BM cells, or purified hematopoietic progenitors were injected. Encapsulated structures were found in the infarcted areas of a large fraction of hearts after injecting MSCs (22 of 43, 51.2%) and unfractionated BM cells (6 of 46, 13.0%). These formations contained calcifications and/or ossifications. In contrast, no pathological abnormalities were found after injection of purified hematopoietic progenitors (0 of 5, 0.0%), fibroblasts (0 of 5, 0.0%), vehicle only (0 of 30, 0.0%), or cytokine-induced mobilization of BM cells (0 of 35, 0.0%). We conclude that the developmental fate of BM-derived cells is not restricted by the surrounding tissue after myocardial infarction and that the MSC fraction underlies the extended bone formation in the infarcted myocardium. These findings seriously question the biologic basis and clinical safety of using whole BM and in particular MSCs to treat nonhematopoietic disorders.