Miniaturized engineered heart tissues from hiPSC-derived triple cell type co-cultures to study human cardiac function.

Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better than standard two-dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 × 104 cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery.

Introduction of a Geminin mScarlet Reporter into H2B-mTurq2 hiPSCs for Live-cell Imaging of Proliferation and Cell Cycling.

We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1-110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.

Generation of LUMCi041-A-2: Equipping a PAX3 reporter iPSC line with doxycycline inducible H2B-mTurquoise2 for live cell imaging.

An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end we introduced a 3rd generation "all-in-one" doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development.

Interpretation of field potentials measured on a multi electrode array in pharmacological toxicity screening on primary and human pluripotent stem cell-derived cardiomyocytes.

Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy.

Human heart disease: lessons from human pluripotent stem cell-derived cardiomyocytes.

Technical advances in generating and phenotyping cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are now driving their wider acceptance as in vitro models to understand human heart disease and discover therapeutic targets that may lead to new compounds for clinical use. Current literature clearly shows that hPSC-CMs recapitulate many molecular, cellular, and functional aspects of human heart pathophysiology and their responses to cardioactive drugs. Here, we provide a comprehensive overview of hPSC-CMs models that have been described to date and highlight their most recent and remarkable contributions to research on cardiovascular diseases and disorders with cardiac traits. We conclude discussing immediate challenges, limitations, and emerging solutions.

Small molecule absorption by PDMS in the context of drug response bioassays.

The polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb small molecules such as drugs. In microfluidic devices like "Organs-on-Chip", designed to examine cell behavior and test the effects of drugs, this might impact drug bioavailability. Here we developed an assay to compare the absorption of a test set of four cardiac drugs by PDMS based on measuring the residual non-absorbed compound by High Pressure Liquid Chromatography (HPLC). We showed that absorption was variable and time dependent and not determined exclusively by hydrophobicity as claimed previously. We demonstrated that two commercially available lipophilic coatings and the presence of cells affected absorption. The use of lipophilic coatings may be useful in preventing small molecule absorption by PDMS.

Follow-up of Thalidomide treatment in patients with Hereditary Haemorrhagic Telangiectasia.

BACKGROUND: Patients with a hereditary vascular disorder called Rendu-Osler-Weber syndrome (Hereditary Haemorrhagic Telangiectasia, HHT) haemorrhage easily due to weak-walled vessels. Haemorrhage in lungs or brain can be fatal but patients suffer most from chronic and prolonged nosebleeds (epistaxis), the frequency and intensity of which increases with age. Several years ago, it was discovered serendipitously that the drug Thalidomide had beneficial effects on the disease symptoms in several of a small group of HHT patients: epistaxis and the incidence of anaemia were reduced and patients required fewer blood transfusions. In addition, they reported a better quality of life. However, Thalidomide has significant negative side effects, including neuropathy and fatigue. METHODS: We followed up all HHT patients in the Netherlands who had been taking Thalidomide at the time the original study was completed to find out (i) how many had continued taking Thalidomide and for how long (ii) the nature and severity of any side-effects and (iii) whether side-effects had influenced their decision to continue taking Thalidomide. RESULTS: Only a minority of patients had continued taking the drug despite its beneficial effects on their symptoms and that the side effects were the primary reason to stop. CONCLUSION: Despite symptom reduction, alternative treatments are still necessary for epistaxis in HHT patients and a large-scale clinical trial is not justified although incidental use in the most severely affected patients can be considered.

Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage, blood outgrowth endothelial cells and cells from milk teeth.

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g., for children and minors) dental pulp cells from milk teeth represent a valuable alternative.

Repolarization reserve determines drug responses in human pluripotent stem cell derived cardiomyocytes.

Unexpected induction of arrhythmias in the heart is still one of the major risks of new drugs despite recent improvements in cardiac safety assays. Here we address this in a novel emerging assay system. Eleven reference compounds were administrated to spontaneously beating clusters of cardiomyocytes from human pluripotent stem cells (hPSC-CM) and the responses determined using multi-electrode arrays. Nine showed clear dose-dependence effects on field potential (FP) duration. Of these, the Ca(2+) channel blockers caused profound shortening of action potentials, whereas the classical hERG blockers, like dofetilide and d,l-sotalol, induced prolongation, as expected. Unexpectedly, two potent blockers of the slow component of the delayed rectifier potassium current (I(Ks)), HMR1556 and JNJ303, had only minor effects on the extracellular FP of wild-type hPSC-CM despite evidence of functional I(Ks) channels. These compounds were therefore re-evaluated under conditions that mimicked reduced "repolarization reserve," a parameter reflecting the capacity of cardiomyocytes to repolarize and a strong risk factor for the development of ventricular arrhythmias. Strikingly, in both pharmacological and genetic models of diminished repolarization reserve, HMR1556 and JNJ03 strongly increased the FP duration. These profound effects indicate that I(Ks) plays an important role in limiting action potential prolongation when repolarization reserve is attenuated. The findings have important clinical implications and indicate that enhanced sensitization to repolarization-prolonging compounds through pharmacotherapy or genetic predisposition should be taken into account when assessing drug safety.

Stem cell self-renewal: lessons from bone marrow, gut and iPS toward clinical applications.

The hematopoietic stem cell (HSC) is the prototype organ-regenerating stem cell (SC), and by far the most studied type of SC in the body. Currently, HSC-based therapy is the only routinely used SC therapy; however, advances in the field of embryonic SCs and induced pluripotent SCs may change this situation. Interest into in vitro generation of HSCs, including signals for HSC expansion and differentiation from these more primitive SCs, as well as advances in other organ-specific SCs, in particular the intestine, provide promising new applications for SC therapies. Here, we review the basic principles of different SC systems, and on the basis of the experience with HSC-based SC therapy, provide recommendations for clinical application of emerging SC technologies.

The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands.

One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.).

Regulation of cardiomyocyte differentiation of embryonic stem cells by extracellular signalling.

Investigating the signalling pathways that regulate heart development is essential if stem cells are to become an effective source of cardiomyocytes that can be used for studying cardiac physiology and pharmacology and eventually developing cell-based therapies for heart repair. Here, we briefly describe current understanding of heart development in vertebrates and review the signalling pathways thought to be involved in cardiomyogenesis in multiple species. We discuss how this might be applied to stem cells currently thought to have cardiomyogenic potential by considering the factors relevant for each differentiation step from the undifferentiated cell to nascent mesoderm, cardiac progenitors and finally a fully determined cardiomyocyte. We focus particularly on how this is being applied to human embryonic stem cells and provide recent examples from both our own work and that of others.

[Freezing umbilical-cord blood and bone marrow for one's own use: present-day quackery?]. / Invriezen van navelstrengbloed en beenmerg voor eigen gebruik: hedendaagse kwakzalverij?

In the Netherlands, the practice of private freezing and banking of umbilical-cord blood is increasing. In a questionnaire, Dutch midwives and gynaecologists were asked about their attitude towards cord-blood collection if asked to perform this after delivery. The response rate was 35% (125/356) and 71% (71/100), respectively. Two-thirds of those asked responded that they would comply. The most common application of cord blood is in the treatment of (malignant) blood disorders. The use of autologous cord blood is, however, often not the best choice for treating leukaemia in young children and the number of stem cells is often too low in a single-cord blood sample to treat older children and adults. Although frequently suggested in the lay press, there is no proven effect in other indications, such as amyotrophic lateral sclerosis, multiple sclerosis and myocardial infarction. Information on therapeutic applications of cord blood from companies with commercial interests is leading to the exploitation ofpregnantwomen. The government should consider limiting this practice and prohibiting the activities of these companies in the Netherlands pending scientific evidence for their claims.

[Progress and clashes in stem cell therapy research]. / Vooruitgang en strubbelingen bij het onderzoek naar stamceltherapie.

The concept of stem cell therapy, the repair of damaged or diseased tissue by transplantation of healthy cells, is deceptively simple. This simplicity has led to the hype among desperate patients, their doctors and the media: it would seem that every ailment can be treated with stem cells. At this time, however, the scientific truth is mostly disappointing. The recent claim from Korea of a major breakthrough - the ability to grow stem cells from cloned embryos - turned out to be fraudulent. With the exception ofapplications in haematologic diseases, skin wounds, and bone and cartilage diseases, most of the putative therapeutic applications of stem cell therapy are still under preclinical investigation. These include the treatment of Parkinson's and Alzheimer's disease, paraplegia, cerebral infarction, amyotrophic lateral sclerosis, multiple sclerosis and muscular dystrophy. The treatment of cardiac infarction is currently being investigated in clinical trials.

New and viable cells to replace lost and malfunctioning myocardial tissue.

The use of stem cells for cardiac repair is a promising opportunity for developing new treatment strategies as the applications are theoretically unlimited and lead to actual cardiac tissue regeneration. Human embryonic stem cells were only recently cloned and their capacity to differentiate into true cardiomyocytes makes them in principle an unlimited source of transplantable cells for cardiac repair, although practical and ethical constraints exist. Also, the study of embryonic stem cells and their differentiation into cardiomyocytes will bring forth new insights into the molecular processes involved in cardiomyocyte-development and -proliferation, which could lead to the development of other strategies to augment in vivo cardiomyocyte numbers. On the other hand, somatic stem cells are alternative cell sources that can be used for cell transplantation purposes. They do not evoke ethical issues and bear less ethical constraints. However, they also appear to be much more restricted in their differentiation potential than the embryonic stem cells. Here we discuss the use of both cell types, embryonic and somatic stem cells, in relation with their importance for the clarification of cardiomyocyte-development and their possible usefulness for clinical therapy.

Smad expression during kidney development.

Signaling by the transforming growth factor (TGF)-beta superfamily is important during kidney development. Here, we describe the spatial and temporal expression patterns of the Smads, the transcription factors that translate TGF- signals into gene expression. RT-PCR data and in situ hybridization analysis showed that the receptor-regulated (R) Smads (Smad1, -2, -3, -5, and -8), the common partner Smad (Smad4), and the inhibitory (I) Smads (Smad6 and -7) were all expressed during mouse kidney development from embryonic day 12 until the end of nephrogenesis at postnatal day 15. Each Smad had a distinct spatial distribution. All were expressed by mesenchymal cells in the nephrogenic zone and were downregulated once these cells began to epithelialize. The common partner Smad, Smad4, was present in uninduced mesenchymal cells and at ureteric bud tips. The bone morphogenetic-responsive R-Smads, Smad1, -5, and -8, were mainly expressed in the nephrogenic zone, whereas the TGF-- responsive R-Smads were predominantly noted in the medullary interstitium. Expression of the I-Smad Smad7 was also seen in mesenchymal cells in the interstitium. Based on the observed patterns of expression, we speculate that individual or combinations of Smads may play specific roles in cell-fate determination during kidney development.

The human adult cardiomyocyte phenotype.

AIM: Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. METHODS: Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. RESULTS: The isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions. CONCLUSION: Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.

[Human embryonic stem cells: possibilities for future cell transplantation therapy]. / Humane embryonale stamcellen: toekomstmogelijkheden voor celtransplantatietherapie.

Human embryonic stem cells are of great importance, and Parkinson's disease is given as an example of a condition that could benefit from the development of stem cell-based transplantation therapies. The reason for this is fairly obvious: the disease is caused by the loss of only one cell type from the brain that has one major function, namely the production of dopamine. Replacement of these cells should in principle cure the disease. But what are stem cells and how far is scientific research from being able to offer stem cell-based therapy in the clinic to patients suffering from Parkinson's disease, and other chronic diseases? These questions are addressed here together with a critical evaluation of short and long-term clinical perspectives, and a discussion of possible alternatives such as adult stem cells.

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice.

Deletion of the transforming growth factor beta1 (TGF-beta1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF-beta more precisely, we inactivated the TGF-beta type I receptor (TbetaRI) gene by gene targeting. Mice lacking TbetaRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TbetaRI(-/-) yolk sacs, clonogenic assays on yolk sac-derived hematopoietic precursors in vitro revealed that TbetaRI(-/-) mice exhibit normal hematopoietic potential compared with wild-type and heterozygous siblings. Endothelial cells derived from TbetaRI-deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TbetaRI is crucial for the function of TGF-beta during vascular development and can not be compensated for by the activin receptor-like kinase-1 (ALK-1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF-beta signaling via TbetaRI.

Transforming growth factor beta and mouse development.

Transforming growth factor beta has many biological effects including the control of cellular growth, differentiation, migration and extracellular matrix production; these are all processes essential for normal development. Although mice first generated more than eight years ago, bearing mutations in TGF beta ligands demonstrated the importance of TGF beta-induced signal transduction pathways for development in mammals but complete functional analysis is still lacking. Here, the current state-of-the-art in mouse development is reviewed. As a basis for understanding function, the principle features of mouse development over the 21 days of pregnancy are described and illustrated, from fertilization of the egg to mid-gestation when organogenesis is largely complete. This is completed with a description of when and where TGF beta ligands, receptors and downstream signalling molecules are expressed as the mouse embryo develops. The functions of TGF beta in preimplantation development, in implantation of the embryos in the uterine wall and in postimplantation development are then described through a review of the literature on gene ablation of the ligands, receptors and downstream molecules, or the ectopic expression of dominant negative forms of the receptors in vivo, which interfere with normal signal transduction. The evidence confirms multifunctional roles at all stages of development.