Optimizing the Design and Analysis of Clinical Trials for Antibacterials Against Multidrug-resistant Organisms: A White Paper From COMBACTE's STAT-Net.

Innovations are urgently required for clinical development of antibacterials against multidrug-resistant organisms. Therefore, a European, public-private working group (STAT-Net; part of Combatting Bacterial Resistance in Europe [COMBACTE]), has reviewed and tested several innovative trials designs and analytical methods for randomized clinical trials, which has resulted in 8 recommendations. The first 3 focus on pharmacokinetic and pharmacodynamic modeling, emphasizing the pertinence of population-based pharmacokinetic models, regulatory procedures for the reassessment of old antibiotics, and rigorous quality improvement. Recommendations 4 and 5 address the need for more sensitive primary end points through the use of rank-based or time-dependent composite end points. Recommendation 6 relates to the applicability of hierarchical nested-trial designs, and the last 2 recommendations propose the incorporation of historical or concomitant trial data through Bayesian methods and/or platform trials. Although not all of these recommendations are directly applicable, they provide a solid, evidence-based approach to develop new, and established, antibacterials and address this public health challenge.

Challenges and solutions for clinical development of new antibacterial agents: results of a survey among pharmaceutical industry professionals.

As the number of antibacterial medicines in the pipeline remains low, we anonymously surveyed pharmaceutical industry professionals on challenges and solutions for clinical development of these agents. Challenges were reported primarily as financial and regulatory. For multidrug-resistant organisms, there are needs for rapid diagnostic tests, new regulatory guidance, and adaptation of endpoints/trial designs. Regulators and public/private initiatives are addressing these challenges to help ensure that proposed solutions have the support of all involved stakeholders.

Fluctuations in phenylalanine concentrations in phenylketonuria: a review of possible relationships with outcomes.

Fluctuations in blood phenylalanine concentrations may be an important determinant of intellectual outcome in patients with early and continuously treated phenylketonuria (PKU). This review evaluates the studies on phenylalanine fluctuations, factors affecting fluctuations, and if stabilizing phenylalanine concentrations affects outcomes, particularly neurocognitive outcome. Electronic literature searches of Embase and PubMed were performed for English-language publications, and the bibliographies of identified publications were also searched. In patients with PKU, phenylalanine concentrations are highest in the morning. Factors that can affect phenylalanine fluctuations include age, diet, timing and dosing of protein substitute and energy intake, dietary adherence, phenylalanine hydroxylase genotype, changes in dietary phenylalanine intake and protein metabolism, illness, and growth rate. Even distribution of phenylalanine-free protein substitute intake throughout 24h may reduce blood phenylalanine fluctuations. Patients responsive to and treated with 6R-tetrahydrobiopterin seem to have less fluctuation in their blood phenylalanine concentrations than controls. An increase in blood phenylalanine concentration may result in increased brain and cerebrospinal fluid phenylalanine concentrations within hours. Although some evidence suggests that stabilization of blood phenylalanine concentrations may have benefits in patients with PKU, more studies are needed to distinguish the effects of blood phenylalanine fluctuations from those of poor metabolic control.

Effect of food on the pharmacokinetics of zoliflodacin granules for oral suspension: Phase I open-label randomized cross-over study in healthy subjects.

Gonorrhea is a sexually transmitted infection for which antibiotic treatment options have declined due to increasing antibiotic resistance. Zoliflodacin, an investigational oral spiropyrimidinetrione antibiotic with activity against Neisseria gonorrhoeae strains that are multidrug-resistant, including to third-generation cephalosporins, is in phase III development for uncomplicated gonorrhea. This phase I, parallel, open-label, randomized, crossover study in healthy adults evaluated the effect of food on the pharmacokinetics of single 3 or 4 g doses of zoliflodacin administered as granules for oral suspension in the fasted state or after consumption of a standardized high-fat meal. Forty-seven out of 48 randomized subjects completed the study. Oral administration of zoliflodacin with food delayed the absorption rate, compared with fasted state, with time to maximum concentration (Tmax ) increasing from 3 to 6 h for the 3 g dose, and 2.5 to 4 h for the 4 g dose, but had no impact on the elimination of zoliflodacin. The maximum concentration (Cmax ) and area under the plasma concentration-time curve from time 0 to 24 h (AUC(0-24) ) significantly increased with food by 52% and 94% for the 3 g dose, and by 89% and 108% for the 4 g dose. Forty-two percent of participants reported a total of 34 treatment-emergent adverse events (TEAEs), which were all considered mild in severity. Headache was the most common TEAE (22/48 subjects, 45.8%) and the only TEAE reported in more than one subject. In conclusion, administration of single 3 and 4 g doses of zoliflodacin as granules for oral suspension, with a high-fat meal was well-tolerated and resulted in statistically significant increases in peak and overall systemic exposure to zoliflodacin.

Plasmodium-induced inflammation by uric acid.

Infection of erythrocytes with the Plasmodium parasite causes the pathologies associated with malaria, which result in at least one million deaths annually. The rupture of infected erythrocytes triggers an inflammatory response, which is induced by parasite-derived factors that still are not fully characterized. Induced secretion of inflammatory cytokines by these factors is considered a major cause of malaria pathogenesis. In particular, the inflammatory cytokine tumor necrosis factor (TNF) is thought to mediate most of the life-threatening pathologies of the disease. Here we describe the molecular characterization of a novel pathway that results in the secretion of TNF by host cells. We found that erythrocytes infected by Plasmodium accumulate high concentrations of hypoxanthine and xanthine. Degradation of Plasmodium-derived hypoxanthine/xanthine results in the formation of uric acid, which triggers the secretion of TNF. Since uric acid is considered a "danger signal" released by dying cells to alert the immune system, Plasmodium appears to have co-evolved to exploit this warning system. Identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease.

Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection.

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.

Dual effect of Plasmodium-infected erythrocytes on dendritic cell maturation.

BACKGROUND: Infection with Plasmodium is the cause of malaria, a disease characterized by a high inflammatory response in the blood. Dendritic cells (DC) participate in both adaptive and innate immune responses, influencing the generation of inflammatory responses. DC can be activated through different receptors, which recognize specific molecules in microbes and induce the maturation of DC. METHODS: Using Plasmodium yoelii, a rodent malaria model, the effect of Plasmodium-infected erythrocytes on DC maturation and TLR responses have been analysed. RESULTS: It was found that intact erythrocytes infected with P. yoelii do not induce maturation of DC unless they are lysed, suggesting that accessibility of parasite inflammatory molecules to their receptors is a key issue in the activation of DC by P. yoelii. This activation is independent of MyD88. It was also observed that pre-incubation of DC with intact P. yoelii-infected erythrocytes inhibits the maturation response of DC to other TLR stimuli. The inhibition of maturation of DC is reversible, parasite-specific and increases with the stage of parasite development, with complete inhibition induced by schizonts (mature infected erythrocytes). Plasmodium yoelii-infected erythrocytes induce a broad inhibitory effect rendering DC non-responsive to ligands for TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9. CONCLUSIONS: Despite the presence of inflammatory molecules within Plasmodium-infected erythrocytes, which are probably responsible for DC maturation induced by lysates, intact Plasmodium-infected erythrocytes induce a general inhibition of TLR responsiveness in DC. The observed effect on DC could play an important role in the pathology and suboptimal immune response observed during the disease. These results help to explain why immune functions are altered during malaria, and provide a system for the identification of a parasite-derived broad inhibitor of TLR-mediated signaling pathways.

Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression.

Abstract It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.

The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation.

Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Down-regulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes.

Stimulating Research and Development of New Antibiotics While Ensuring Sustainable Use and Access: Further Insights from the DRIVE-AB Project and Others.

Global discussions are ongoing on how to stimulate antibiotic research and development in order to provide patients with new antibiotics able to address the challenges of antimicrobial resistance. In this supplement, we present nine articles derived from the research performed as part of the Innovative Medicine Initiative-funded DRIVE-AB project and others. These publications provide new evidence and arguments in the debate around economic incentives to stimulate antibiotic innovation, including characteristics, implementation and governance.

Appropriate endpoints for evaluation of new antibiotic therapies for severe infections: a perspective from COMBACTE's STAT-Net.

PURPOSE: In this era of rising antimicrobial resistance, slowly refilling antibiotic development pipelines, and an aging population, we need to ensure that randomized clinical trials (RCTs) determine the added benefit of new antibiotic agents effectively and in a valid way, especially for severely ill patients. Unfortunately, universally accepted endpoints for the evaluation of new drugs in severe infections are lacking. METHODS: We review and discuss the current practices and challenges regarding endpoints in RCTs in this field and propose novel approaches. RESULTS: Usual endpoints actually recommended for drug development suffer from important flaws. Mortality requires large sample size and only partly related to the infectious process. Clinical cure rate is highly subjective in critically ill patients where symptoms may be related to other intercurrent events. Currently, composite endpoints, hierarchical nested designs, and competing risks analysis seem to be the most promising new tools for designing and analyzing clinical trials in this area, although they require further validation. CONCLUSION: Regulatory authorities, pharmaceutical companies, and clinicians need to agree on the most appropriate clinical endpoints for severe infections to ensure efficient approval of new, effective antibiotic agents.

Development of new antibiotics: taking off finally?

Since 2010, awareness of the global threat caused by antimicrobial resistance (AMR) has risen considerably and multiple policy and research initiatives have been implemented. Research and development (R&D) of much-needed new antibiotics active against multiresistant pathogens is a key component of all programmes aiming at fighting AMR, but it has been lagging behind owing to scientific, regulatory and economic challenges. Although a few new antibiotics might be available in Switzerland in the next 5 years, these new agents are not based on new mechanisms of action and are not necessarily active against resistant pathogens for which there is the highest unmet medical need, i.e. multiresistant Gram-negative bacteria. Of the three new antibiotics with pending authorisation in Switzerland for systemic treatment of severe infections, oritavancin and tedizolid target Gram-positive pathogens, while only ceftolozane+tazobactam partially covers multiresistant Gram-negative pathogens. Among six antibiotics currently in phase III of clinical development, delafloxacin and solithromycin will also be useful mostly for Gram-positive infections. Importantly, the four other compounds are active against multiresistant Gram-negative pathogens: ceftazidime+avibactam, meropenem+RPX7009, eravacycline and plazomicin. The three last compounds are also active against carbapenem-resistant Enterobacteriaceae (CRE). A few compounds active against such pathogens are currently in earlier clinical development, but their number may decrease, considering the risk of failure over the course of clinical development. At last, through public and political awareness of pathogens with high public health impact and unmet medical need, development of innovative economic incentives and updated regulatory guidance, R&D of new antibiotics is slowly taking off again.

Development and psychometric validation of measures to assess the impact of phenylketonuria and its dietary treatment on patients' and parents' quality of life: the phenylketonuria - quality of life (PKU-QOL) questionnaires.

BACKGROUND: The aim of our study was to develop and validate the first set of PKU-specific Health-related Quality of Life (HRQoL) questionnaires that: 1) were developed for patients with PKU and their parents, 2) cover the physical, emotional, and social impacts of PKU and its treatment on patients' lives, 3) are age specific (Child PKU-QOL, Adolescent PKU-QOL, Adult PKU-QOL), 4) enable the evaluation of the HRQoL of children by their parents (Parent PKU-QOL), and 5) have been cross-culturally adapted for use in seven countries (i.e. France, Germany, Italy, The Netherlands, Spain, Turkey and the UK). METHODS: The PKU-QOL questionnaires were developed according to reference methods including patients', parents' and healthcare professionals' interviews; testing in a pilot study (qualitative step in six countries), and linguistic validation of the finalised pilot versions in Turkish. For finalisation and psychometric validation, the pilot versions were included in a multicentre, prospective, non-interventional, observational study conducted in 34 sites in France, Germany, Italy, The Netherlands, Spain, Turkey and the UK. Iterative multi-trait analyses were conducted. Psychometric properties were assessed (concurrent and clinical validity, internal consistency reliability and test-retest reliability). RESULTS: Data from 559 subjects (306 patients, 253 parents) were analysed. After finalisation, the PKU-QOL questionnaires included 40 items (Child PKU-QOL), 58 items (Adolescent PKU-QOL), 65 items (Adult PKU-QOL) and 54 items (Parent PKU-QOL), distributed in four modules: PKU symptoms, PKU in general, administration of Phe-free protein supplements and dietary protein restriction. The measurement properties of the Adolescent, Adult and Parent PKU-QOL questionnaires were overall fairly satisfactory, but weaker for the Child questionnaire. CONCLUSIONS: The four PKU-QOL questionnaires developed for different ages (Child PKU-QOL, Adolescent PKU-QOL, Adult PKU-QOL), and for parents of children with PKU (Parent PKU-QOL) are valid and reliable instruments for assessing the multifaceted impact of PKU on patients of different age groups (children, adolescents and adults) and their parents, and are available for use in seven countries. They are very promising tools to explore how patients' perceptions evolve with age, to increase knowledge of the impact of PKU on patients and parents in different countries, and to help monitor the effect of therapeutic strategies.

Assessment of the impact of phenylketonuria and its treatment on quality of life of patients and parents from seven European countries.

BACKGROUND: The strict and demanding dietary treatment and mild cognitive abnormalities seen in PKU treated from a young age can be expected to affect the health-related quality of life (HRQoL) of patients and their families. Our aim was to describe the HRQoL of patients with PKU from a large international study, using generic HRQoL measures and an innovative PKU-specific HRQoL questionnaire (PKU-QOL). Analyses were exploratory, performed post-hoc on data collected primarily to validate the PKU-QOL. METHODS: A multicentre, prospective, non-interventional, observational study conducted in France, Germany, Italy, The Netherlands, Spain, Turkey and the UK. Patients diagnosed with PKU aged ≥9 years old and treated with a Phe-restricted diet and/or Phe-free amino acid protein supplements and/or pharmacological therapy were included in the study; parents of at least one patient with PKU aged <18 years were also included. HRQoL was assessed by generic measures (Pediatric Quality-of-Life Inventory; Medical Outcome Survey 36 item Short Form; Child Health Questionnaire 28 item Parent Form) and the newly developed PKU-QOL. Mean generic domain scores were interpreted using published reference values from the general population. PKU-QOL domain scores were described overall and in different subgroups of patients defined according to severity of PKU, overall assessment of patient's health status by the investigator and treatment with tetrahydrobiopterin (BH4). RESULTS: Data from 559 subjects were analysed: 306 patients (92 children, 110 adolescents, 104 adults) and 253 parents. Mean domain scores of generic measures in the study were comparable to the general population. The highest PKU-QOL impact scores (indicating greater impact) were for emotional impact of PKU, anxiety about blood Phe levels, guilt regarding poor adherence to dietary restrictions or Phe-free amino acid supplement intake and anxiety regarding blood Phe levels during pregnancy. Patients with mild/moderate PKU and those receiving BH4 reported lower practical and emotional impacts of the diet and Phe-free amino acid supplement intake. CONCLUSION: Patients with PKU showed good HRQoL in the study, both with the generic and PKU-specific measures. Negative impacts of PKU on a patient's life, including the emotional impact of PKU and its management, was delineated by the PKU-QOLs across all age groups.

Embryonic stem cells: new possible therapy for degenerative diseases that affect elderly people.

The capacity of embryonic stem (ES) cells for virtually unlimited self renewal and differentiation has opened up the prospect of widespread applications in biomedical research and regenerative medicine. The use of these cells would overcome the problems of donor tissue shortage and implant rejection, if the cells are made immunocompatible with the recipient. Since the derivation in 1998 of human ES cell lines from preimplantation embryos, considerable research is centered on their biology, on how differentiation can be encouraged toward particular cell lineages, and also on the means to enrich and purify derivative cell types. In addition, ES cells may be used as an in vitro system not only to study cell differentiation but also to evaluate the effects of new drugs and the identification of genes as potential therapeutic targets. This review will summarize what is known about animal and human ES cells with particular emphasis on their application in four animal models of human diseases. Present studies of mouse ES cell transplantation reveal encouraging results but also technical barriers that have to be overcome before clinical trials can be considered.

Improved treatment of multidrug-resistant bacterial infections: utility of clinical studies.

In a time of increasing antibacterial resistance and limited availability of new antibiotics, clinical studies are much needed to assess treatment options against multidrug-resistant organisms (MDROs). In this review, we describe the clinical challenge caused by MDROs and present recent evidence on how clinical studies may generate quality data to improve antibiotic treatment of MDRO infections. To this aim, we critically assess the current status, gaps and challenges associated with observational and interventional studies performed to assess MDRO treatment options. We address why observational studies are useful, which treatment options for MDRO have been explored by observational studies and how to improve quality and usefulness of observational studies. Furthermore, the utility of clinical pharmacokinetic/pharmacodynamic studies for improving MDRO treatment is described. Finally, we discuss interventional study designs, end points and margins, as well as ethical, logistic and statistical challenges, and current regulatory changes proposed to foster the development of new antibiotics.

Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening.

The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC(50): 54, 190 and 23 nM, respectively). Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti-T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC(50) values of 2 nM (PCH6 and CX2). These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis.

Expression and function of alpha-smooth muscle actin during embryonic-stem-cell-derived cardiomyocyte differentiation.

Three alpha-muscle actin isoforms are sequentially expressed during in vivo cardiac development. alpha-Smooth muscle actin is first and transiently expressed, followed by alpha-skeletal and finally alpha-cardiac actin. The significance of these transitions in actin gene expression during myogenesis remains to be determined. To understand whether actin isoforms have specific functions during cardiac development and cardiomyocyte contractility, we have hampered alpha-smooth muscle and alpha-skeletal actin expression and organization during embryonic stem cell differentiation towards cardiomyocyte. We show that the sequence of actin isoform expression displays similar pattern in the in vitro model and in mouse heart embryogenesis. Treatment with an interfering fusion peptide containing the N-terminal sequence of alpha-smooth muscle actin during a time window preceding spontaneous beating, prevents proper cardiac sarcomyogenesis, whereas alpha-skeletal actin-fusion peptide has no effect. Knockdown of alpha-smooth muscle actin in embryonic stem cells using RNA interference also affects cardiac differentiation. The application of both fusion peptides on beating embryoid bodies impairs frequency. These results suggest specific functional activities for actin isoforms in cardiogenesis and cardiomyocyte contractility.

Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: a molecular and electrophysiological approach.

Cardiomyocytes derived from human embryonic stem cells constitute a promising cell source for the regeneration of damaged hearts. The assessment of their in vitro functional properties is mandatory to envisage appropriate cardiac cell-based therapies. In this study, we characterized human embryonic stem cell-derived cardiomyocytes over a 3-month period, using patch-clamp or intracellular recordings to assess their functional maturation and reverse transcriptase-polymerase chain reaction to evaluate the expression of ion channel-encoding subunits. I(to1) and I(K1), the transient outward and inward rectifier potassium currents, were present in cardiomyocytes only, whereas the rapid delayed rectifier potassium current (I(Kr)), pacemaker current (I(f)), and L-type calcium current (I(Ca,L)) could be recorded both in undifferentiated human embryonic stem cells and in cardiomyocytes. Most of the currents underwent developmental maturation in cardiomyocytes, as assessed by modifications in current density (I(to1), I(K1), and I(Ca,L)) and properties (I(f)). Ion-channel mRNAs were always present when the current was recorded. Intracellular recordings in spontaneously beating clusters of cardiomyocytes revealed changes in action potential parameters and in response to pharmacological tools according to time of differentiation. In summary, human embryonic stem cell-derived cardiomyocytes mature over time during in vitro differentiation, approaching an adult phenotype. Disclosure of potential conflicts of interest is found at the end of this article.

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells.

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.