Eugen Kolossov

Host-Dependent Tumorigenesis of Embryonic Stem Cell Transplantation in Experimental Stroke

The therapeutical potential of transplantation of undifferentiated and predifferentiated murine embryonic stem cells for the regeneration of the injured brain was investigated in two rodent stroke models. Undifferentiated embryonic stem cells xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury migrated along the corpus callosum towards the damaged tissue and differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. The authors demonstrated a hitherto unrecognized inverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells, which raises concerns about safety provisions when the therapeutical potential of human embryonic stem cells is tested in preclinical animal models.

Cardiac specific differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells may represent an alternative source of functionally intact cardiomyocytes for the causal treatment of cardiovascular diseases. However, this requires cardiac-specific differentiation of stem cells and the selection of pure lineages consisting of early embryonic cardiomyocytes. Therefore, an understanding of the basic mechanisms of heart development is essential for selective differentiation of embryonic stem cells into cardiac cells. The development of cardiac cells from embryonic stem cells is regulated by several soluble factors and signalling molecules together with cardiac specific transcription factors such as the zinc-finger GATA proteins and Nkx-2.5. GATA-4 and Nkx-2.5 seem to be essential for heart development. The use of enhanced green fluorescent protein (EGFP) under the control of cardiac-specific promoters in combination with the ES cell system has allowed for the functional characterisation of cardiac precursor cells. Embryonic stem cell-derived cardiomyocytes developmentally express similar cardiac-specific proteins, ion channels and signalling molecules to that of adult cardiomyocytes. Furthermore, identification of growth factors and signalling molecules under cell culture conditions is crucial for the selective cardiac differentiation of embryonic stem cells. Therefore, serum-free culture conditions have to be established in order to examine the influence of different growth factors and signalling molecules on cardiac development and/or formation from ES cells. Although significant progress has been made in generating cardiac cell lineage by the combination of genetically manipulative methods with selective culture conditions for cell transplantation therapy, one of the remaining future challenges for transplantation in humans is the immunological rejection of the engrafted cardiomyocytes.

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 &bgr;-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.

Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes

The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the α‐myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP‐positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single‐cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch‐clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker‐like cardiomyocytes. In accordance, multielectrode‐array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP‐positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the α‐myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.

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.

Cardiac specific expression of the green fluorescent protein during early murine embryonic development

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac α‐actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac α‐actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.

Activity of complex III of the mitochondrial electron transport chain is essential for early heart muscle cell differentiation

During development of the heart, mitochondria proliferate within cardiomyocytes. It is unclear whether this is a response to the increasing energy demand or whether it is part of the developmental program. To investigate the role of the electron transport chain (ETC) in this process, we used transgenic murine embryonic stem (ES) cells in which the green fluorescent protein gene is under control of the a‐myosin heavy chain promoter (a‐MHC), allowing easy monitoring of cardiomyocyte differentiation. Spontaneous contraction of these cells within embryoid bodies (EBs) was not affected by inhibition of the ETC, suggesting that early heart cell function is sufficiently supported by anaerobic ATP production. However, heart cell development was completely blocked when adding antimycin A, an inhibitor of ETC complex III, before initiation of differentiation, whereas KCN did not block differentiation, strongly suggesting that specifically complex III function rather than mitochondrial ATP production is necessary for early heart cell development. When the underlying mechanism was examined, we noticed that antimycin A but not KCN lead to inhibition of spontaneous intracellular Ca++ oscillations, whereas both substances decreased mitochondrial membrane potential, as expected. We postulate that mitochondrial complex III activity is necessary for these Ca++ oscillations, which in turn are a prerequisite for cardiomyocyte differentiation.

Cellular cardiomyoplasty in a transgenic mouse model.

BACKGROUND Recent progress in the cardiotypic differentiation of embryonic and somatic stem cells opens novel prospects for the treatment of cardiovascular disorders. The aim of the present study was to develop a novel surgical approach that allows standardized cellular cardiomyoplasty in mouse with low-perioperative mortality. METHODS Reproducible transmural lesions were generated by cryoinjury followed by intramural injection of embryonic cardiomyocytes using a newly designed holding device and vital dye staining. This approach was validated with a transgenic mouse model, in which the live reporter gene-enhanced green fluorescent protein (EGFP) is under control of a cardiac-specific promoter. RESULTS The perioperative mortality was 10%. The engrafted EGFP-positive cardiomyocytes could be identified in a high percentage (72.2%, n=36) of operated animals. CONCLUSIONS This novel approach enables reliable cellular replacement therapy in mouse and greatly facilitates the analysis of its molecular, cellular, and functional efficacy.

The use of quantitative image analysis in the assessment of in vitro embryotoxicity endpoints based on a novel embryonic stem cell clone with endoderm-related GFP expression

The capacity of pluripotent embryonic stem cells (ESC) to differentiate in vitro into various tissues provides the opportunity to develop an in vitro assay for investigating mechanisms of developmental toxicity. ESC clones carrying tissue specific reporter gene constructs are currently being developed. The clones should allow the quantification of the effects of chemicals on the development of germ layers and main target tissues. We report the establishment of the alpha-fetoprotein_GFP/D3 reporter gene clone: alpha-fetoprotein (AFP) enhancers and the homologous promoter regulate green fluorescent protein (GFP) expression in cells of the D3-ESC clone. AFP was used as a marker for endodermal cells. Differentiation of this clone via embryoid bodies (EBs, spheroids of cells) leads to green fluorescence on the surfaces of EBs. AFP- related GFP expression was confirmed. An easy and quick image analysis-based endpoint measurement was developed for quantifying low amounts of cells expressing GFP. As demonstrated with the embryotoxic chemical diphenylhydantoin, image analysis can be used to distinguish between a general effect on EB growth and a specific effect on the development of GFP-positive endodermal cells. Endoderm development was inhibited at a different dose than cardiomyocyte development.

Differential Role of bFGF and VEGF for Vasculogenesis

Primary vascular plexus originate from angioblasts through a process called vasculogenesis. The precise role of basic fibroblast growth factor (bFGF) and the vascular endothelial growth factor (VEGF) are both suggested as key regulators in vasculogenesis is still unclear. This crucial aspect was investigated by using time lapse observation of in vitro generated embryonic stem (ES) cell-derived endothelial structures which were recognizable by using the platelet cell adhesion molecule (PECAM-1) driven endothelial-specific expression of the live reporter gene enhanced green fluorescent protein (EGFP). In serum free conditions VEGF led to improved survival of angioblasts and to the formation of primitive endothelial tubes whereas bFGF alone increased their survival. Our study suggests that the complex process of vasculogenesis can be driven by VEGF alone but not by bFGF.