The Biological and Ethical Basis of the Use of Human Embryonic Stem Cells for In Vitro Test Systems or Cell Therapy

Human embryonic stem cells (hESC) are now routinely cultured in many laboratories, and differentiation protocols are available to generate a large variety of cell types. In an ongoing ethical debate opinions of different groups are based on varying sets of religious, historical, cultural and scientific arguments as well as on widely differing levels of general information. We here give an overview of the biological background for non-specialists, and address all issues of the current stem cell debate that are of concern in different cultures and states. Thirty-five chapters address embryo definition, potential killing and the beginning of human life, in addition to matters of human dignity, patenting, commercialisation, and potential alternatives for the future, such as induced pluripotent (reprogrammed) stem cells. All arguments are compiled in a synopsis, and compromise solutions, e.g. for the definition of the beginning of personhood and for assigning dignity to embryos, are suggested. Until recently, the major application of hESC was thought to be transplantation of cells derived from hESC for therapeutic use. We discuss here that the most likely immediate uses will rather be in vitro test systems and disease models. Major and minor pharmaceutical companies have entered this field, and the European Union is sponsoring academic research into hESC-based innovative test systems. This development is supported by new testing strategies in Europe and the USA focussing on human cell-based in vitro systems for safety evaluations, and shifting the focus of toxicology away from classical animal experiments towards a more mechanistic understanding.JRC.I.3-In-vitro method

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives

<p>Abstract</p> <p>Background</p> <p>Mouse embryonic stem (ES) cells remain pluripotent <it>in vitro </it>when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency.</p> <p>Results</p> <p>By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [<it>Pluri </it>genes], ii) LIF induced genes [<it>Lifind </it>genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of <it>Pluri </it>and <it>Lifind </it>genes, and revealed their differential modulation by <it>Oct4 </it>or <it>Nanog </it>master genes. Individual knock down of a selection of <it>Pluri </it>and <it>Lifind </it>genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed.</p> <p>Conclusion</p> <p>We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.</p

Extracellular Ca2+ Sensing in C-Cells and Parathyroid Cells

An essential function of C-cells and parathyroid cells is to monitor the extracellular Ca2+ concentration. The Ca2+-dependent secretion of calcitonin (CT) and parathyroid hormone is known to be mediated by corresponding changes in the intracellular Ca2+ concentration. To address the question of whether Ca2+ influx through voltage-dependent Ca2+ channels couples the extracellular to the intracellular Ca2+, we applied the patch clamp technique to C-cells of the rMTC 44-2 cell line and to parathyroid cells of the PT-r cell line. The rMTC cells displayed dihydropyridine-sensitive, voltage-dependent, high-threshold Ca2+ channels which allowed ion influx even at the resting potential of about -40 mV. Increases of the concentration of the extracellular divalent cation or adding the Ca2+ channel agonist Bay K 8644 stimulated the steady state ion influx. In contrast, PT-r cells exhibited only fast inactivating, low-threshold Ca2+ channel currents with no steady state conductivity for Ca2+ at the resting potential of around -40 mV. We conclude that dihydropyridine-sensitive Ca2+ channels allow steady state transmembranous Ca2+ influx in C-cells, thereby increasing the cytosolic Ca2+ and CT secretion. Parathyroid cells, however, lack long-lasting Ca2+ channel currents and obviously sense the extracellular Ca2+ concentration by other mechanisms

Fusion and regenerative therapies: is immortality really recessive?

Harnessing cellular fusion as a potential tool for regenerative therapy has been under tentative investigation for decades. A look back the history of fusion experiments in gerontology reveals that whereas some studies indicate that aging-related changes are conserved in fused cells, others have demonstrated that fusion can be used as a tool to revoke cellular senescence and induce tissue regeneration. Recent findings about the role of fusion processes in tissue homeostasis, replenishment, and repair link insights from fusion studies of previous decades with modern developments in stem cell biology and regenerative medicine. We suggest that age-associated loss of regenerative capacity is associated with a decline of effectiveness in stem cell fusion. We project how studies into the fusion of stem cells with tissue cells, or the fusion between activator stem cells and patient cells might help in the development of applications that "rejuvenate" certain target cells, thereby strategically reinstating a regeneration cascade. The outlook is concluded with a discussion of the next research milestones and the potential hazards of fusion therapies. © 2007 Mary Ann Liebert, Inc

Fusion and regenerative therapies: is immortality really recessive?

Harnessing cellular fusion as a potential tool for regenerative therapy has been under tentative investigation for decades. A look back the history of fusion experiments in gerontology reveals that whereas some studies indicate that aging-related changes are conserved in fused cells, others have demonstrated that fusion can be used as a tool to revoke cellular senescence and induce tissue regeneration. Recent findings about the role of fusion processes in tissue homeostasis, replenishment, and repair link insights from fusion studies of previous decades with modern developments in stem cell biology and regenerative medicine. We suggest that age-associated loss of regenerative capacity is associated with a decline of effectiveness in stem cell fusion. We project how studies into the fusion of stem cells with tissue cells, or the fusion between activator stem cells and patient cells might help in the development of applications that "rejuvenate" certain target cells, thereby strategically reinstating a regeneration cascade. The outlook is concluded with a discussion of the next research milestones and the potential hazards of fusion therapies. © 2007 Mary Ann Liebert, Inc

Modeling genetic cardiac channelopathies using induced pluripotent stem cells - Status quo from an electrophysiological perspective

Long QT syndrome (LQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT) are genetic diseases of the heart caused by mutations in specific cardiac ion channels and are characterized by paroxysmal arrhythmias, which can deteriorate into ventricular fibrillation. In LQTS3 and BrS different mutations in the SCN5A gene lead to a gain-or a loss-of-function of the voltage-gated sodium channel Nav1.5, respectively. Although sharing the same gene mutation, these syndromes are characterized by different clinical manifestations and functional perturbations and in some cases even present an overlapping clinical phenotype. Several studies have shown that Na(+ )current abnormalities in LQTS3 and BrS can also cause Ca2+-signaling aberrancies in cardiomyocytes (CMs). Abnormal Ca2+ homeostasis is also the main feature of CPVT which is mostly caused by heterozygous mutations in the RyR2 gene. Large numbers of disease-causing mutations were identified in RyR2 and SCN5A but it is not clear how different variants in the SCN5A gene produce different clinical syndromes and if in CPVT Ca2+ abnormalities and drug sensitivities vary depending on the mutation site in the RyR2. These questions can now be addressed by using patient-specific in vitro models of these diseases based on induced pluripotent stem cells (iPSCs). In this review, we summarize different insights gained from these models with a focus on electrophysiological perturbations caused by different ion channel mutations and discuss how will this knowledge help develop better stratification and more efficient personalized therapies for these patients

Low concentrations of ethanol but not of dimethyl sulfoxide (DMSO) impair reciprocal retinal signal transduction

The model of the isolated and superfused retina provides the opportunity to test drugs and toxins. Some chemicals have to be applied using low concentrations of organic solvents as carriers. Recently, E-/R-type (Ca(v)2.3) and T-type (Ca(v)3.2) voltage-gated Ca2+ channels were identified as participating in reciprocal inhibitory retinal signaling. Their participation is apparent, when low concentrations of NiCl2 (15 mu M) are applied during superfusion leading to an increase of the ERG b-wave amplitude, which is explained by a reduction of amacrine GABA-release onto bipolar neurons. During these investigations, differences were observed for the solvent carrier used. Recording of the transretinal receptor potentials from the isolated bovine retina. The pretreatment of bovine retina with 0.01 % (v/v) dimethylsulfoxide did not impair the NiCl2-mediated increase of the b-wave amplitude, which was 1.31-fold +/- 0.03 of initial value (n = 4). However, pretreatment of the retina with the same concentration of ethanol impaired reciprocal signaling (0.96-fold +/- 0.05, n = 4). Further, the implicit time of the b-wave was increased, suggesting that ethanol itself but not DMSO may antagonize GABA-receptors. Ethanol itself but not DMSO may block GABA receptors and cause an amplitude increase by itself, so that reciprocal signaling is impaired

MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes

An in depth investigation at the genomic level is needed to identify early human-relevant cardiotoxicity biomarkers that are induced by drugs and environmental toxicants. The main objective of this study was to investigate the role of microRNAs (miRNAs) as cardiotoxicity biomarkers using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) that were exposed to doxorubicin (DOX) as a gold standard cardiotoxicant. hiPSC-CMs were exposed to 156 nM DOX for 2 days or for 6 days of repeated exposure, followed by drug washout and incubation in drug-free culture medium up to day 14 after the onset of exposure. The induced miRNAs were profiled using miRNA microarrays, and the analysis of the data was performed using the miRWalk 2.0 and DAVID bioinformatics tools. DOX induced early deregulation of 14 miRNAs (10 up-regulated and 4 down-regulated) and persistent up-regulation of 5 miRNAs during drug washout. Computational miRNA gene target predictions suggested that several DOX-responsive miRNAs might regulate the mRNA expression of genes involved in cardiac contractile function. The hiPSC-CMs exposed to DOX in a range from 39 to 156 nM did not show a significant release of the cytotoxicity marker lactate dehydrogenase (LDH) compared to controls. Quantitative real-time PCR analyses confirmed the early deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303, and also the prolonged up-regulation of miR-182-5p, miR-4423-3p and miR-34c-5p. Thus, we identified and validated miRNAs showing differential DOX-responsive expression before the occurrence of cytotoxicity markers such as LDH, and these miRNAs also demonstrated the significant involvement in heart failure in patients and animal models. These results suggest that the DOX-induced deregulated miRNAs in human CMs may be used as early sensitive cardiotoxicity biomarkers for screening potential drugs and environmental cardiotoxicants with a similar mechanism of action

Surgical Approaches in Psychiatry: A Survey of the World Literature on Psychosurgery

Brain surgery to promote behavioral or affective changes in humans remains one of the most controversial topics at the interface of medicine, psychiatry, neuroscience, and bioethics. Rapid expansion of neuropsychiatric deep brain stimulation has recently revived the field and careful appraisal of its 2 sides is warranted: namely, the promise to help severely devastated patients on the one hand and the dangers of premature application without appropriate justification on the other. Here, we reconstruct the vivid history of the field and examine its present status to delineate the progression from crude freehand operations into a multidisciplinary treatment of last resort. This goal is accomplished by a detailed reassessment of numerous case reports and small-scale open or controlled trials in their historical and social context. The different surgical approaches, their rationale, and their scientific merit are discussed in a manner comprehensible to readers lacking extensive knowledge of neurosurgery or psychiatry, yet with sufficient documentation to provide a useful resource for practitioners in the field and those wishing to pursue the topic further

Co-transplantation of Mesenchymal Stromal Cells and Induced Pluripotent Stem Cell-Derived Cardiomyocytes Improves Cardiac Function After Myocardial Damage

Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) represent an attractive resource for cardiac regeneration. However, survival and functional integration of transplanted iPS-CM is poor and remains a major challenge for the development of effective therapies. We hypothesized that paracrine effects of co-transplanted mesenchymal stromal cells (MSCs) augment the retention and therapeutic efficacy of iPS-CM in a mouse model of myocardial infarction (MI). To test this, either iPS-CM, MSC, or both cell types were transplanted into the cryoinfarction border zone of syngeneic mice immediately after injury. Bioluminescence imaging (BLI) of iPS-CM did not confirm enhanced retention by co-application of MSC during the 28-day follow-up period. However, histological analyses of hearts 28 days after cell transplantation showed that MSC increased the fraction of animals with detectable iPS-CM by 2-fold. Cardiac MRI analyses showed that from day 14 after transplantation on, the animals that have received cells had a significantly higher left ventricular ejection fraction (LVEF) compared to the placebo group. There was no statistically significant difference in LVEF between animals transplanted only with iPS-CM or only with MSC. However, combined iPS-CM and MSC transplantation resulted in higher LVEF compared to transplantation of single-cell populations during the whole observation period. Histological analyses revealed that MSC increased the capillarization in the myocardium when transplanted alone or with iPS-CM and decreased the infarct scar area only when transplanted in combination with iPS-CM. These results indicate that co-transplantation of iPS-CM and MSC improves cardiac regeneration after cardiac damage, demonstrating the potential of combining multiple cell types for increasing the efficacy of future cardiac cell therapies