The quality of QSAR models: problems and solutions.

Assessment of the quality of goodness-of-fit and the confidence in predictivity (prediction power) are the main terms used to define the statistical quality of QSAR models. Three parts of this assessment can be defined as: (1) Measure of goodness-of-fit. (2) Validation of model stability. (3) Predictivity analysis. Currently there are no mandatory requirements for the validation methods to be used and rules for the quantitative confidence estimates. To compare the statistical quality of QSAR models it is necessary to have an overall statistical quality index which will depend on the goodness-of-fit, validation and predictivity results together. To do so it is necessary to define the set of mandatory parameters for all three parts of assessment listed above and develop the approach for overall quality estimates based on these parameters. It is also necessary to include into the overall index the penalty mechanism for parameter absence. The goal of the present study is to analyse parameters for all three parts of the QSAR model statistical quality assessment and investigate the flexible weighting approach for the overall statistical quality index development. Due the different statistical parameters traditionally used for assessment of goodness-of-fit it is necessary to create the mechanism, which allows flexible set of parameters to be used for the overall statistical quality index. Only after approval by scientific community and regulatory boards the final set of mandatory parameters can be selected.

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 alpha-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 alpha-myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.

Implication of therapeutic cloning for organ transplantation.

Replacement of damaged myocardium with electrically functional, contracting syncytium with a balanced blood supply remains a key goal for the treatment of hearts damaged by coronary heart disease or other disorders. Stem cell therapy offers a potential solution. This paper describes the value of in vitro stem cell research to unravel the roles of key regulatory molecules in embryogenesis of myocardium and blood vessels. Studies have shown that functioning myocytes can be derived from stem cells in vitro and engrafted into infarcted areas of heart where they develop into functional adult like cardiomyocytes with action potentials and capacity for beta adrenergic and muscarinic regulation. Further studies have identified specific roles for platelet endothelial cell adhesion molecule (PECAM), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in the sequential differentiation of blood vessels and capillaries.

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.

Establishment of an in vitro reporter gene assay for developmental cardiac toxicity.

This study is based on the unique potential of pluripotent embryonic stem (ES) cells to differentiate in vitro into embryoid bodies containing cell lineages representative of most cell types found in the mammalian fetus. However, the use of wild type ES cells as an in vitro assay for embryotoxicological studies is complicated by the simultaneous development of various cellular phenotypes. This prevents a quantitative assessment of drug effects on one specific cell type. Here we report the effects of 15 chemicals on cardiac differentiation as determined by various specific toxicological endpoints such as morphological inspection (contractile activity), quantitative mRNA analysis and cardiac-specific expression of green fluorescent protein (GFP), used as a quantitative reporter. The data from the different endpoints have been subjected to a statistical analysis, and a preliminary prediction model is proposed. The results demonstrate that genetically-engineered ES cells could provide a valuable tool for estimating the developmental cardiotoxic potential of compounds in vitro and form the basis for automated analysis in a high-throughput system.

Differentiation of green fluorescent protein-labeled embryonic stem cell-derived neural precursor cells into Thy-1-positive neurons and glia after transplantation into adult rat striatum.

OBJECT: The aim of this investigation was to assess new information concerning the capacity of transplanted embryonic stem cell (ESC)-derived neuronal cells to migrate into host brain and to evaluate these cells as a possible source for cell replacement therapy in neurodegenerative disorders such as Parkinson's disease (PD). METHODS: The authors investigated the ability of ESC-derived neural precursor cells to migrate and differentiate in a host striatum by using a D3-derived ESC clone that was transfected stably with a chicken beta-actin cytomegalovirus enhancer-driven green fluorescent protein (GFP)-labeled construct. This procedure allowed easy monitoring of all transplanted cells because of the green fluorescent labeling of donor cells. This approach also afforded easy estimation of cell integration and simultaneous observation of the entire transplanted cell population in relation to immunocytochemically identified neuronal and glial differentiation. After selection of nestin-positive neural precursor cells in a synthetic medium, they were implanted into the striatum of male adult Wistar rats. Their integration was analyzed on morphological studies performed 3 days to 4 weeks posttransplantation. CONCLUSIONS: The investigators found that after transplantation, a subpopulation of GFP-labeled cells differentiated into various neural morphological types that were positive for the mouse-specific Thy-1 antigen, which is known be expressed on neurons, as well as being positive for the astroglial marker glial fibrillary acidic protein. Moreover, GFP-expressing cells that were negative for either of these markers remained close to the injection site, presumably representing other derivatives of the neural lineage. Together, these findings contribute to basic research regarding future transplantation strategies in neurodegenerative diseases such as PD.

Establishment of ionic channels and signalling cascades in the embryonic stem cell-derived primitive endoderm and cardiovascular system.

The first organ system to be established in early embryogenesis is the cardiovascular system which develops upon interaction with hypoblastic cells of the primitive endoderm. Here we focus on recent work on embryoid bodies derived from pluripotent embryonic stem (ES) cells. Ca(2+) oscillations and Ca(2+) signalling pathways during the differentiation of primitive endodermal cell layers are reported. Furthermore, the development-dependent expression of ion channels and the buildup of signalling cascades involved in the modulation of voltage-dependent L-type Ca(2+) channels during early cardiomyogenesis and the formation of functional vascular structures in the process of vasculogenesis and angiogenesis are reviewed. We also report on the use of green fluorescent protein reporter gene expression under the control of cardiac-specific promoters, e.g. the human cardiac alpha-actin promoter, which enables the identification and in vivo characterization of cardiomyocytes at very early stages of cardiomyogenesis.

Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

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 alpha-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 alpha-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.