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Introduction: The Croatian Association for Diabetes and Metabolic Disorders of the Croatian Medical Association has issued in 2011 the first national guidelines for the nutrition, education, self-control, and pharmacotherapy of diabetes type 2. According to the increased number of available medicines and new evidence related to the effectiveness and safety of medicines already involved in the therapy there was a need for update of the existing guidelines for the pharmacotherapy of type 2 diabetes in the Republic of Croatia., Participants: as co-authors of the Guidelines there are listed all members of the Croatian Association for Diabetes and Metabolic Diseases, as well as other representatives of professional societies within the Croatian Medical Association, who have contributed with comments and suggestions to the development of the Guidelines., Evidence: These guidelines are evidence-based, according to the GRADE system (eng. Grading of Recommendations, Assessment, Development and Evaluation), which describes the level of evidence and strength of recommendations., Conclusions: An individual patient approach based on physiological principles in blood glucose control is essential for diabetes' patients management. Glycemic targets and selection of the pharmacological agents should be tailored to the patient, taking into account the age, duration of disease, life expectancy, risk of hypoglyce- mia, comorbidities, developed vascular and other complications as well as other factors. Because of all this, is of national interest to have a practical, rational and applicable guidelines for the pharmacotherapy of type 2 diabetes.

The objective of the study was to examine the association between fish and shellfish intake and diabetes in an island population, and the design of the study was Cross-sectional. Two independent population-based field surveys were conducted in Hvar Island of the eastern Adriatic coast of Croatia in May 2007 and May 2008, with a total of 1,379 adult participants. In multivariable logistic regression models, total fish intake was positively associated with diabetes prevalence in the total population (OR(Q4 vs. Q1) = 1.64; 95% CI = 1.01-2.66; p-trend = 0.09). Oily fish intake also exhibited a positive association with diabetes prevalence in the total population (OR(Q4 vs. Q1) = 2.22; 95% CI = 1.35-3.64; p-trend = 0.01) and in analyses stratified by body mass index, males and those with a high waist circumference. The study suggests an association between oily fish intake and diabetes in the population of the Hvar Island in Croatia. Longitudinal studies incorporating measures of persistent organic pollutants and local cooking practices are warranted to identify factors in fatty fish that may influence the development or persistence of diabetes.

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

Chronic granulomatous disease (CGD) is an inherited orphan disorder caused by mutations in one of the five genes encoding reduced nicotinamide-adenine-dinucleotide-phosphate oxidase subunits, which subsequently lead to impairment in the production of microbicidal reactive oxygen species (ROS). In order to offer several cell line models of CGD and therefore support research on pathophysiology and new therapeutic approaches, we optimized protocols to differentiate induced pluripotent stem cells (iPSCs) from wild-type, X(0)-, AR22(0)- and AR47(0)-CGD patient's fibroblasts into neutrophils and into macrophages. Aberrant genetic clones were discarded after chromosome karyotyping and array-comparative genomic hybridization analysis. All remaining iPSC lines showed human embryonic stem cell-like morphology, expressed all tested pluripotency markers and formed embryoid bodies that contained cells originating from all three primary germ layers. Furthermore, each CGD patient-specific iPSC line retained the gp91 (phox) , p47 (phox) , and p22 (phox) mutations found in the corresponding patient's neutrophils. The average production of CD34(+) progenitors was of 1.5×10(6) cells after 10 days of differentiation of 10×10(6) iPSCs. They were terminally differentiated into about 3×10(5) neutrophils or into 3×10(7) macrophages. Based on morphological, phenotypical, and functional criteria both phagocyte types were mature and indistinguishable from the native human neutrophils and macrophages. However, neutrophils and macrophages derived from X(0)-, AR22(0)-, and AR47(0)-CGD patient-specific iPSC lines lacked ROS production and the corresponding mutated proteins. To simplify the phagocytes' production upon request, progenitors can be cryopreserved. In conclusion, we describe a reproducible, simple, and efficient way to generate neutrophils and macrophages from iPSCs and provide a new cellular model for the AR22(0)-CGD genetic form that has not been described before.

Cell loss after transplantation is a major limitation for cell replacement approaches in regenerative medicine. To assess the survival kinetics of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) we generated transgenic murine iPSC lines which, in addition to CM-specific expression of puromycin N-acetyl-transferase and enhanced green fluorescent protein (EGFP), also constitutively express firefly luciferase (FLuc) for bioluminescence (BL) in vivo imaging. While undifferentiated iPSC lines generated by random integration of the transgene into the genome retained stable FLuc activity over many passages, the BL signal intensity was strongly decreased in purified iPS-CM compared to undifferentiated iPSC. Targeted integration of FLuc-expression cassette into the ROSA26 genomic locus using zinc finger nuclease (ZFN) technology strongly reduced transgene silencing in iPS-CM, leading to a several-fold higher BL compared to iPS-CM expressing FLuc from random genomic loci. To investigate the survival kinetics of iPS-CM in vivo, purified CM obtained from iPSC lines expressing FLuc from a random or the ROSA26 locus were transplanted into cryoinfarcted hearts of syngeneic mice. Engraftment of viable cells was monitored by BL imaging over 4 weeks. Transplanted iPS-CM were poorly retained in the myocardium independently of the cell line used. However, up to 8% of cells survived for 28 days at the site of injection, which was confirmed by immunohistological detection of EGFP-positive iPS-CM in the host tissue. Transplantation of iPS-CM did not affect the scar formation or capillary density in the periinfarct region of host myocardium. This report is the first to determine the survival kinetics of drug-selected iPS-CM in the infarcted heart using BL imaging and demonstrates that transgene silencing in the course of iPSC differentiation can be greatly reduced by employing genome editing technology. FLuc-expressing iPS-CM generated in this study will enable further studies to reduce their loss, increase long-term survival and functional integration upon transplantation.

Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns-particularly of DNAm patterns associated with tissue type and aging., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

High mtDNA variation in Southeastern Europe (SEE) is a reflection of the turbulent and complex demographic history of this area, influenced by gene flow from various parts of Eurasia and a long history of intermixing. Our results of 1035 samples (488 from Croatia, 239 from Bosnia and 130 from Herzegovina, reported earlier, and 97 Slovenians and 81 individuals from Žumberak, reported here for the first time) show that the SEE maternal genetic diversity fits within a broader European maternal genetic landscape. The study also shows that the population of Žumberak, located in the continental part of Croatia, developed some unique mtDNA haplotypes and elevated haplogroup frequencies due to distinctive demographic history and can be considered a moderate genetic isolate. We also report seven samples from the Bosnian population and one Herzegovinian sample designated as X2* individuals that could not be assigned to any of its sublineages (X2a'o) according to the existing X2 phylogeny. In an attempt to clarify the phylogeny of our X2 samples, their mitochondrial DNA has been completely sequenced. We suppose that these lineages are signs of local microdifferentiation processes that occurred in the recent demographic past in this area and could possibly be marked as SEE-specific X2 sublineages., (© 2014 John Wiley & Sons Ltd/University College London.)

Introduction: The development of new antiarrhythmic agents is challenging and is hampered by high attrition rate of novel drug candidates. One of the reasons for this is limited predictability of existing preclinical models for drug assessment. Cardiomyocytes (CMs) derived from disease-specific induced pluripotent stem cells (iPSC) represent a novel in vitro cellular model of cardiac arrhythmias with an unprecedented potential for generating new mechanistic insight into disease pathophysiology and improving the process of drug development., Areas Covered: This review outlines recent studies demonstrating the suitability and limitations of iPSC-derived CMs (iPS-CMs) for in vitro modeling inherited arrhythmias and drug testing. The authors focus on channelopathies and outline the properties of iPS-CMs, highlighting their utility and limitations for investigating the mechanism of cardiac arrhythmias and drug discovery., Expert Opinion: The iPS-CMs represent a valuable addition to the already existing armamentarium of cardiac arrhythmic models. However, the superiority of iPS-CMs over other arrhythmia models has not yet been rigorously established and the limitations of the model must be overcome before its full potential for antiarrhythmic drug discovery can be realized. Nevertheless, iPS cell-based platforms hold a great potential for increasing our knowledge about cellular arrhythmia mechanisms and improving the drug discovery process.

Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na(+) channel in the LQTS-3 CM. This hints at a defective Na(+) channel caused by deficiency in open-state inactivation of the Na(+) channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability.

Chronic complications of diabetes are the main cause of mortality and disability in people with diabetes, while also leading to growing healthcare system cost burdens. In order to analyze the effects of possible interventions on the health of population and their effectiveness in reducing the cost of the healthcare system, we have made health-economic models of diabetes chronic complications development. The models simulated long-term effect of improving glycemic control, blood pressure and lipid status in patients over a period of 10 years. The simulation results showed that the total savings over the analyzed period could amount to over 2 billion HRK, as effective long term control of risk factors leads to a reduction in the development of complications and death in people with diabetes. Successful treatments of patients provide a variety of savings for each of the chronic complications, and are obtained by simulating the biggest savings in the cost of hospitalization and rehabilitation. The cost-effectiveness analysis leads to the conclusion that the more intensive patient's control and earlier application of a treatment is cost-effective, and change of the healthcare system activities is a necessity.

Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca(2+) channels of different cell types. Moreover, influx of Ca(2+) through L-type Ca(2+) channels (LTCCs) activates Ca(2+) signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca(2+) transient and reduction of LTCCs current density (I(CaL)) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca(2+) channel α(1C) (Ca(V)1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca(2+) concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

Background/aims: Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells., Methods: The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence., Results: Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to β-adrenergic and muscarinic stimulations., Conclusion: Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells., (Copyright © 2013 S. Karger AG, Basel.)

Metabolic syndrome presents a significant public health problem today, in Croatia as well as the rest of the world, considering the fact that the World Health Organization classifies its diagnostic criteria, such as high blood pressure and obesity, among ten major risk factors for health. The research of the prevalence of metabolic syndrome among the inhabitants of the island of Cres included a total of 385 adult subjects, 249 women and 136 men. The incidence of metabolic syndrome in two subpopulations of the inhabitants: the inhabitants of the town of Cres (urban population) and the inhabitants of other settlements (rural population), was analysed. The incidence of metabolic syndrome among the inhabitants of the island of Cres was determined according to definitions of the WHO (World Health Organization) and the NCEP-ATP III program (National Cholesterol Education Program-Third Adult Treatment Panel III). It was established that the overall prevalence of metabolic syndrome on the island of Cres was 14% according to the WHO definition (20.6% in men and 10.4% in women) and 24.9% according to the NCEP-ATP III definition (33.1% in men and 20.5% in women). The study also showed that the prevalence of risk factors for developing metabolic syndrome and complex diseases was much higher among the subjects from other settlements than among the subjects from the town of Cres, a consequence of age and a lifestyle with lack of physical activity.

This study presents genetic diversity and structure of contemporary Krk islanders revealed by high-resolution mitochondrial DNA analysis on a sample of 132 unrelated autochthonous adults from seven different settlements and regions of the island. Relatively high level of haplogroup and haplotype diversity in the overall island sample is an indicator of numerous migrations and gene flows throughout the history. Expectedly, the results show the highest frequency of haplogroup H (33.3%), yet this value is much lower compared to different Croatian and other European mainland populations. An interesting finding refers to highly elevated frequencies of some haplogroups, otherwise rare in Croatia and most of the Europe, such as I (11.3%) and W (7.6%) in Krk population, especially pronounced in some settlements. At the level of settlements, many of the major European haplogroups were found to be absent from their mtDNA gene pools, whereas several others show a pronounced deviation from an average. Overall, our results suggest a tangled interplay of different evolutionary forces, such as founder effects and a few strong bottlenecks, presumably due to epidemics, which have occurred in various periods of the island's history. Cultural customs, such as frequent endogamy in some regions of the island during past centuries, have additionally shaped its genetic structure into the observed present-day diversity patterns.

Transplantation of purified pluripotent stem cell-derived cardiomyocytes into damaged myocardium might become a therapy to improve contractile function after myocardial infarction. However, engraftment remains problematic. Aim of this study was to investigate whether murine embryonic fibroblasts (MEFs) support the functional integration of purified embryonic stem cell-derived cardiomyocytes (ES-CMs). Neonatal murine ventricular tissue slices were subjected to oxygen and glucose deprivation to simulate irreversible ischemia. Vital tissue slices served as control. Vital and avital tissue slices were cultured with or without MEFs before coculturing with clusters of puromycin-selected ES-CMs. Integration of ES-CM clusters was assessed morphologically, motility by long-term microscopy, and functional integration by isometric force measurements. We observed a good morphological integration into vital but a poor integration into avital slices. Adding MEFs improved morphological integration into irreversibly damaged slices and enabled purified ES-CMs to migrate and to confer force. We conclude that noncardiomyocytes like MEFs support morphological integration and force transmission of purified ES-CMs by enabling adhesion and migration.

There is growing interest in the use of cardiomyocytes purified from embryonic stem (ES) cells for tissue engineering and cardiomyoplasty. However, most transplanted cells are lost shortly after transplantation due to the lack of integration into the host tissue and subsequent apoptosis. Here we examine whether murine embryonic fibroblasts (MEFs) can support the integration of purified murine ES cell-derived cardiomyocytes in a 3-dimensional tissue culture model based on a freezed-dryed collagen matrix with tubular structure. Collagen matrix was seeded either with cardiomyocytes alone or in combination with MEFs. The collagen sponges that were transplanted with cardiomyocytes alone showed neither morphological nor functional integration of viable cells. Cardiomyocytes also did not appear to be capable of attaching quantitatively to any of 16 different 2-dimensional biomaterials. However, cardiomyocytes co-cultured with MEFs formed fiber-like structures of rod-shaped cells with organized sarcomeric structure that contracted spontaneously. Electrical coupling between cardiomyocytes was suggested by strong expression of connexin 43. In addition, MEFs as well as cardiac fibroblasts supported re-aggregation of dissociated cardiomyocytes in hanging drops in the absence of collagen matrix. We conclude that fibroblasts promote cardiomyocyte engraftment and formation of functional 3-dimensional tissue in vitro. Elucidation of the mechanism of this phenomenon may help improve the integration of cardiomyocytes in vivo.

Background: Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells., Results: Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes., Conclusions: These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC-CMs) and iPS cells (iPS-CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5-7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force-frequency relationship (FFR), effects of Ca(2+), Ni(2+), nifedipine, ryanodine, beta-adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC-CMs and iPS-CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 microM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC(50) values were similar. Contractile properties of iPS-CMs and ESC-CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the beta-adrenergic response of iPS-CMs and ESC-CMs.

The transplantation of cardiomyocytes derived from embryonic stem (ES) cells into infarcted heart has been shown to improve heart function in animal models. However, immune rejection of transplanted cells may hamper the clinical application of this approach. Natural killer (NK) cells could play an important role in this process in both autologous and allogeneic settings by eliminating cells expressing low levels of major histocompatibility complex (MHC) class I molecules. Here we characterize embryonic stem cell-derived cardiomyocytes (ESCM) in terms of their sensitivity to NK cells. We show that despite expression of very low levels of MHC class I molecules, murine ESCM were neither recognized nor lysed by activated syngeneic NK cells in vitro. In contrast, undifferentiated ES cells expressing similarly low levels of MHC class I molecules as ESCM were recognized and lysed by NK cells. This differential susceptibility results from the differential expression of ligands for the major activating natural killer cell receptor natural-killer group 2 member D (NKG2D) and intercellular adhesion molecule 1 (ICAM-1) on ES cells versus ESCM. NKG2D ligands and ICAM-1 were expressed on ES cells but were absent from ESCM. Undifferentiated ES cells were lysed by NK cells in a perforin-dependent manner. However, simultaneous blockade of NKG2D and ICAM-1 by antibodies inhibited this killing. These data suggest that in the course of differentiation ESCM acquire resistance to NK cell-mediated lysis by downregulating the expression of ligands required for activation of NK cell cytotoxicity.

Aims: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells., Methods and Results: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to beta-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation., Conclusions: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty., (2009 S. Karger AG, Basel.)

Replacement of diseased tissues with healthy cells derived from embryonic stem (ES) cells has a potential to become, in the future, a better alternative to current treatments of a number of conditions characterized by irreversible tissue injury, such as heart and liver failure, diabetes mellitus and neurodegeneration. However, several obstacles have to be overcome before this new therapeutic modality becomes part of a standard clinical practice. First of all, ethical and safety issues have to be resolved, the methodologies must be developed to enable obtaining sufficient amounts of differentiated cells, and the immune rejection of allogeneic cells must be prevented in order to ensure their long-term engraftment and function. Data on immunological properties of human and murine ES cells and their differentiated derivatives are controversial, ranging from those claiming unique immune-privileged properties for ES cells to those which refute these conclusions. This indicates that much more research is required to definitively understand the immunological features and engraftment capacity of ES cell derivatives. We review here the current state of the art in this new and exciting field of ES cell immunology and discuss the implications of these findings for the development of ES cell-based therapies., ((c) 2008 S. Karger AG, Basel.)

Highly ordered degradation of cell proteins by the ubiquitin-proteasome system, a sophisticated cellular proteolytic machinery, has been identified as a key regulatory mechanism in many eukaryotic cells. Accumulating evidence reveals that the ubiquitin-proteasome system is involved in the regulation of fundamental processes in mammalian stem and progenitor cells of embryonic, neural, hematopoietic, and mesenchymal origin. Such processes, including development, survival, differentiation, lineage commitment, migration, and homing, are directly controlled by the ubiquitin-proteasome system, either via proteolytic degradation of key regulatory proteins of signaling and gene expression pathways or via nonproteolytic mechanisms involving the proteasome itself or posttranslational modifications of target proteins by ubiquitin or other ubiquitin-like modifiers. Future characterization of the precise roles and functions of the ubiquitin-proteasome system in mammalian stem and early progenitor cells will improve our understanding of stem cell biology and may provide an experimental basis for the development of novel therapeutic strategies in regenerative medicine. Disclosure of potential conflicts of interest is found at the end of this article.

Using quantitative RT-PCR, we found that T1 rat prostate cancer cell relative FGF-1 transcript content was about 180-fold greater than that of FGF-2. This difference in transcript content was not representative of T1 cell relative FGF-1 and FGF-2 protein content which showed, at most, only a 4- to 5-fold greater FGF-1 content. Testosterone caused time-dependent down-regulation of prostate cancer cell FGF-2 transcript content without influencing either FGF-1 or FGF-8 transcript content or T1 cell proliferation. Moreover, testosterone-mediated down-regulation of prostate cancer cell FGF-2 transcripts did not result in a statistically significant change in 21.5 or 17.0 kD FGF-2 isoform content. By contrast, an approximately 20% statistically significant decrement in 19.5 kD FGF-2 isoform content was demonstrable following 24 h testosterone treatment. However, following 72 h testosterone treatment, T1 cell 19.5 kD FGF-2 isoform content was not statistically significantly different from that of control. It is probable that the modest and variable decrement in 19.5 kD isoform content is not physiologically significant and is attributable to artifact resulting from difficulty quantifying this minor component of the FGF-2 isoforms. Transient transfection analysis showed that androgen caused concentration-dependent increases in MMTV-LTR regulated expression of chloramphenicol acetyl transferase activity. Consequently, the failure of androgen to affect either T1 cell FGF-1 and FGF-8 transcript content or T1 cell proliferation could not be attributed to defective androgen receptor function. Moreover, the absence of a close relationship between T1 cell FGF-2 transcript and FGF-2 protein content implies that FGF-2 transcript content is not the dominant determinant of prostate cancer cell FGF-2 protein content. Testosterone-mediated down-regulation of prostate-cancer-cell gene expression may have significance for clinical management of human disease that is treated by androgen ablation. The possibility that such ablation may enhance aggressiveness of "androgen-independent" cells by selective upregulation of gene expression merits further consideration.

We constructed expression vectors containing either rat fibroblast growth factor (FGF)-1 for FGF-2 cDNA cloned in either the sense orientation or antisense orientation relative to the metallothionein promoter of plasmid pMTneo.1. Stable AXC/SSh rat prostate cancer cell transfectants expressing either chimeric FGF-1-sense, chimeric FGF-1-antisense, or chimeric FGF-2-antisense transcripts were obtained. Stable transfectants expressing chimeric FGF-2-sense transcripts were not obtained. Control, sense, and antisense transfectants expressed endogenous FGF-1 and endogenous FGF-2 transcripts, implying that transfection did not eliminate endogenous FGF transcripts. Control transfectants and sense transfectants contained FGF-1 isoforms having a mass of 16.4 or 17.3 kDa and FGF-2 isoforms having a mass of 17, 19.5, or 21.5 kDa. Significantly, adult AXC/SSh rat prostate contained only the 17.3 kDa FGF-1 isoform and the 17 kDa FGF-2 isoform, indicating that neoplastic transformation was associated with elaboration of novel, prostate epithelial cell-derived FGF-2 isoforms. FGF-1 antisense RNA expression eliminated transfectant FGF-1 isoforms without affecting FGF-2 isoform content. Similarly, FGF-2-antisense RNA expression eliminated the transfectant 21.5 kDa FGF-2 isoform, diminished the 19.5 kDa FGF-2 isoform content, and reduced the 17 kDa FGF-2 isoform content to barely detectable levels without affecting the FGF-1 isoform content. This established that FGF-antisense RNAs specifically inhibited translation of cognate, endogenous FGF transcripts. Doubling times of control transfectants and sense transfectants were indistinguishable and were not affected by including FGF-1 or FGF-2 in the culture medium. Doubling times of FGF-1-antisense or FGF-2-antisense transfectants were 1.3- to 1.4-fold greater than those of control transfectants or sense transfectants, and either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling times to values indistinguishable from those of control transfectants or sense transfectants. This established that with regard to prostate cancer cell proliferation: (a) endogenous FGF-1 cannot substitute for endogenous FGF-2 eliminated by FGF-2-antisense RNA expression; and (b) endogenous FGF-2 cannot substitute for endogenous FGF-1 eliminated by FGF-1-antisense RNA expression. In contrast, either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling time. The results of these studies establish that endogenous FGF-1 and endogenous FGF-2 modulate prostate cancer cell proliferation and imply that FGF-1 and FGF-2 of endogenous and exogenous origin conjointly control aspects of prostate cancer cell homeostasis. Our findings suggest complex interaction between components of prostate cancer cell regulatory processes and endogenously produced and exogenously accessible FGF-1 and FGF-2.