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iForum Meeting: Europe

October 11, 2016

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Introducing the iForum™ 2016 Meeting: Europe, a full day event for users of iPSC-derived cells. The iForum 2016 Meeting provided a unique opportunity to connect with thought leaders from academia, government, biotech and pharma to discover new applications and uses for differentiated iPS cells. The meeting included innovative scientific talks and poster sessions, as well as an evening event to relax with colleagues.

Posters Presented at the 2016 iForum Europe

101 Human Pluripotent Stem Cells: Tools to Understand Duchenne Myopathies and Develop Therapeutic Approaches
Virginie Mournetas, I-Stem Institute
Abstract
Duchenne and Becker Muscular Dystrophies (DMD and BMD) are recessive X-linked monogenic myopathies caused by mutations in the dystrophin gene. The difference between DMD and BMD is the phenotype severity linked to the dystrophin expression. BMD patients have a dystrophin expression abnormal in quantity and/or size while DMD patients have no dystrophin. DMD has a worldwide prevalence around 1/21,000 boys. Symptoms appear around 2-5 years old with a progressive loss of muscle strength leading to premature death due to cardiac and respiratory failures around 30. There is no treatment available yet to stop the progression of the disease. Our strategy is to use human pluripotent stem cells (hPSCs) to model DMD in a dish. These cells can self-renew or differentiate in vitro into all body's cell types. Our laboratory has developed an easy, fast and robust cell platform using derivatives of hPSCs from healthy and DMD patients expressing high level of 1) embryonic dystrophin Dp412e in mesoderm precursors (3 days of differentiation) or 2) muscular dystrophin Dp427m in skeletal muscle precursors (17 days of differentiation). This platform is used for studying DMD onset and myogenesis and developing novel therapies or improving current ones. As a proof of concept, we are working on exon skipping strategies by the use of a high-throughput screening of PMOs/small molecules combination. We are also setting up high-throughput RNA sequencing to progress our understanding of the disease.
102  New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation
Pierre-Antoine Faye, EA 6309 Maintenance Myelin and Neuropathy Peripheral
Abstract
Induced pluripotent stem cells (hiPSC) are a very useful solution to create and observe the behavior of specific and usually inaccessible cells, such as human motor neurons. Obtained from a patient biopsy by reprograming dermal fibroblasts (DF), hiPSC present the same properties as embryonic stem cells and can generate any cell type after several weeks of differentiation. Today, there are numerous protocols that aim to control hiPSC differentiation. The principal challenge is to obtain a sufficiently enriched specific cell population to study disease pathophysiology and to provide a good model for further investigation and drug screening. The differentiation process is very costly and time-consuming because many specific factors and different culture media must be used. In this study, we used sedimentation field flow fractionation (SdFFF) to prepare enriched populations derived from hiPSC after only 10 days of culture in a classical medium. Based on phenotypic and proteomic characterization, "hyperlayer" elution resulted in a fraction expressing markers of endothelial progenitors while another fraction expressed markers of neural progenitors. The isolation of subpopulations representing various differentiation lineages is of major interest for the production of specialized, cell-enriched fractions and in the preparation of increasingly complex models for the development of new therapeutic tools.
103  Analysis of Different Biomaterials as Scaffolds in Bone Tissue Engineering
Chernet Woyimo Woju, Defence University
Abstract
In tissue engineering for larger bone defects, the use of mesenchymal stem cells has to be combined with the use of scaffold materials. The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders in human and veterinary medicine. However, the safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluated two biomaterials: B30 (70% silica+ 30% collagen) and B30Sr20 (50% silica+ 30% collagen, 20% strontium), a biocompatible composite polymer, as a scaffold for adipose mesenchymal stem cells (MSCs) for application in bone tissue engineering. The quality of biomaterials was measured by analyzing cell adhesion (DAPI staining) and cell proliferation (MTT assay) in conjunction with the scaffold materials. Live cell imaging for cell viability (cell migration) after plating of the materials with cells in culture dishes was done using florescence microscopy. Cell morphology on the biomaterials was investigated using scanning electron microscopy. Furthermore, osteogenic differentiations were tested by driving the cells into the osteogenic lineage using specific growth and differentiation factors. Osteogenic differentiation were analysed by detecting typical morphological changes, by specific staining procedures (Alizarin red method) as well as by using molecular biological techniques.
104  The Mislocalisation of BEST1 in iPSC-RPE Cells Derived from a Family with Autosomal Dominant Vitreoretinochoroidopathy
Amanda-Jayne Carr, University College London
Abstract
Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare, early onset retinal dystrophy that affects the peripheral retina and eye development. ADVIRC is caused by mutations in bestrophin 1 (BEST1), a transmembrane protein thought to function as an ion channel in retinal pigment epithelial (RPE) cells. Previous studies have suggested that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. We created induced pluripotent stem cells (iPSC) from an ADVIRC patient family expressing the c.704T->C (p.V235A) BEST1 missense mutation. Patient and control iPSCs were differentiated into RPE by removal of bFGF from the culture medium. Pigmented foci were isolated from the iPSC cultures by manual dissection and seeded to form a monolayer of RPE. ADVIRC iPSC-RPE cells were pigmented, polarised and formed a monolayer with the classic cobblestone-like appearance. The cells expressed a panel of RPE cell markers, including BEST1, at the RNA and protein level. We found no evidence of alternate splicing of the BEST1 transcript 1 as a result of the ADVIRC mutation; however, immunocytochemical analysis demonstrates that p.V235A missense alteration results in mislocalisation of BEST1 within the RPE cell. These data suggest that the mislocalised expression of BEST1 within the RPE may lead to the distinct clinical phenotype observed in ADVIRC patients.
105  Electric Field Stimulation (EFS) of Human iPSC-derived Neurons Using Hamamatsu FDSS/μCELL
Jean Marc d'Angelo, Hamamatsu Photonics France
Abstract
Hamamatsu developed a 96-channel electrode array system that is mounted on the FDSS/μCELL. It adds electric field stimulations (EFS) to all 96 wells in a microplate simultaneously while fluorescence/luminescence signals are monitored. Using this instrumental setup, we electrically stimulated human iPSC-derived neurons (iCell Neurons) to evoke a Ca2+ response, transient increase of intracellular Ca2+ concentration, which was measured with calcium sensitive fluorescent dyes.
106  Use of iPSCs-based Neural Models to Evaluate CNF1 Therapeutic Potential and Neurotoxicity in the Treatment of Some Intellectual Disability Disorders
Andrea Colarusso, University of Naples Federico II
Abstract
Several studies suggest that modulation of brain Rho GTPases by the bacterial toxin CNF1 reverses the neural damage of CNS disorders like Rett syndrome, Parkinson's disease, and Alzheimer's disease (De Filippis et al., 2015; Musilli et al., 2016; Loizzo et al., 2013). Hence, our research group is trying to develop toxin variants capable of crossing the BBB in order to achieve a protein therapeutic that might be systemically administered. So far, we managed to demonstrate that these protein variants preserve their biological activity. Yet, most of the results reported until now are based either on in vitro simplified systems or animal models. Moreover, CNF1 is reported to be a virulence factor produced by meningitis inducing E. coli strains, but its role in the pathology is not fully clear (Boyer, 2016). As a result, an estimation of benefits and risks is urgent to evaluate CNF1 therapeutic potentiality. These issues may be addressed integrating our current systems with iPSCs-based in vitro models of both normal and pathological neuronal networks. This strategy would allow us: 1) to assess CNF1 therapeutic efficacy on human cells directly obtained from patients, 2) to define the BBB-crossing ability of our CNF1 variants, as in precise conditions as iPSCs-derived endothelial cells show stronger BBB features in comparison to standard BBB models (Lippmann et al., 2012), and 3) to evaluate CNF1 neurotoxicity in normal iPSCs-based neuronal models with a particular attention to BBB integrity.
107  Functional Characterization of iCell Neuronal Networks Using High-resolution CMOS-MEAs
Hayder Amin, Italian Institute of Technology (IIT)
Abstract
The recent availability of human induced pluripotent stem cells (hiPSCs) holds great promise as a novel source of human-derived neurons for cell and tissue therapies as well as for in vitro drug screenings and toxicology that can potentially support the refinement and eventual replacement of animal models. However, there is still a considerable lack of knowledge on the electrophysiological and functional properties of networks formed by these human-derived neurons, thus limiting their application. Upon optimization of cell culture protocols, we demonstrate that both spontaneous and evoked electrical spiking activities of iCell neuronal networks can be characterized over three months on-chip by taking advantage of the resolution provided by CMOS-MEAs. Our results show that responses to electrical stimulation consists in a physiological repertoire of spike waveforms after three months of cell culture, a period of time during which the network undergoes the expression of developing patterns of spontaneous spiking activity. In addition, we demonstrate the impact on the network formation and on the emerging network-wide functional properties, neuronal maturation and synapses stabilization induced by different biochemical substrates for cell adhesion. Taken together, this is a first step toward the development of functional pre-clinical assays to test pharmaceutical compounds on human-derived neuronal networks using CMOS-MEAs.
108  Assessment of Positive and Negative Inotropic Compounds Using an Impedance-based System with Human iPSC-derived Cardiomyocytes under Controlled Pacing Conditions
Xiaoyu Zhang, ACEA Biosciences
Abstract
Understanding drug-mediated modulation of cardiac contractility is an important question from both a therapeutic as well as a safety/toxicity angle. While one of the primary applications for which human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being evaluated is for assessment of pro-arrhythmic compounds (cpds), we wanted to understand if these cells can also be used for assessment of inotropic cpds that modulate contractility. The impedance-based cardiac contractility assay has been systematically studied and validated previously (Scott et al., 2014). While this assay showed comparable sensitivity and specificity using hiPSC-CMs to the well-validated optical-based contractility assay using adult dog cardiomyocytes (Harmer et al., 2012), the assay was unable to delineate inotropic effects from chronotropic effects. In order to address this limitation, we utilized xCELLigence CardioECR system, which combines impedance measurement and electrical pacing, to evaluate the effect of cardio-modulating cpds under pacing & non-pacing conditions. Our data clearly demonstrate: 1) hiPSC-CMs possess negative beating rate and amplitude (the amplitude of impedance waveform) relationship and 2) modulators of cardiac contractility either increased or decreased beating amplitude depending on their respective mechanism of actions. Furthermore, based on our data, the positive inotropic effects of cardio-modulating cpds on beating amplitude could be better assessed under controlled pacing conditions.

109  Pharmacological Evaluation of the Influence of Cell Density on Proarrhythmic Properties of Pharmacological Tool Compounds on a High Throughput Micro Electrode Array Platform
Stefanie Küchler, University of Tübingen
Abstract
hiPSC cardiomyocyte recordings on MEAs have been used as a valuable tool to detect drug induced modulations of the field potential. In addition to prolongations of the field potential by potassium channel antagonists, proarrhythmic events and arrhythmia have been observed. The assay system is currently validated as a regulatory tool in safety pharmacology by the CiPA study. Here we investigate the influence of plating density, exposure time and culture conditions on the pharmacological profile of iCell Cardiomyocytes2 (Cellular Dynamics International).
110  High-resolution Microelectrode Array Platform for Characterizing iPSC-derived Cells at Subcellular, Cellular, and Network Levels
Michele Fiscella, ETH Zurich
Abstract
Microelectrode array (MEA) technology enables fast and high-throughput recording of cellular electrical signals. MEAs are currently used for phenotype characterization and drug toxicity/efficacy testing in iPSC-derived neurons and cardiomyocytes. Here we present a high-resolution microelectronics-based MEA featuring 26400 microelectrodes within a sensing area of 3.85 × 2.10 mm2. The electrodes are arranged in a grid-like configuration with a center-to-center pitch of 17.5 µm, yielding an electrode density of 3265 microelectrodes per mm2. Each electrode or electrode subset can be used to electrically stimulate or record the signals of virtually any cell on the array. The readout noise in the action-potential signal band (300 Hz-10 kHz) is 2.4 µVrms. Cell signals are amplified, band-pass filtered, and digitalized on-chip. We demonstrate that the electrical activity of every cell in the network can be monitored and studied. Furthermore, the high-resolution MEA allows for recording of subcellular details, such as the propagation of action potentials along single axons. In summary, the high-resolution MEA system can extract additional parameters from iPSC-derived cells, which can be used as potential novel biomarkers for phenotype screening and drug testing.
111  Generation of iPS Cells to Model Neurodegenerative Disorders: An Activity from the Cell Culture Core Facility at the Brain and Spine Institute (ICM)
Stephanie Bigou, Brain and Spine Institute (ICM)
Abstract
In recent years, the induced pluripotent stem cell (iPSC) technology has emerged as a powerful tool for the generation of disease-relevant models for the investigation of the underlying molecular and cellular mechanisms, the design of cell-based replacement therapies and the discovery of new treatments. Acquiring this technology in order to promote forefront basic and preclinical research programs in the field was a key objective of the Brain and Spine Institute (ICM). To meet this objective, we benefited from a framework program for translational medicine, the so-called "Instituts Hospitalo-Universitaires" (IHU). The work package devoted to cell culture activities served, in part, to equip an iPSC facility and recruit dedicated technical staff members. Starting in January 2013, we offer different services to researchers: 1) Generation of human iPSC with vectors based on replication-incompetent Sendai vectors to safely deliver reprogramming factors into human healthy fibroblasts, 2) Molecular and functional characterization of iPSC clones; 3) Generation of iPSC-derived neural stem cells and 4) Genetic engineering of iPSC with the CRISPR/Cas9 technology (in progress). Today, the core facility continuously innovates to offer new tools and methodologies for internal research teams, external academic teams, teams from the bioincubator and industrial partners.
112  Utilising Human Derived Induced Pluripotent Cells (iPSC) for Organ Specific Microtissue Formation and High Content Screening
Stephanie Ravenscroft, Cyprotex Discovery
Abstract
Improvements in our current preclinical testing are essential for the discovery of safe and efficient drugs. Human derived induced pluripotent stem cells (iPSC) provide species relevant organ specific in vitro cell models with minimal batch variation and amenable to multiple in vitro applications including 3D cell culture. We have developed novel iPSC-derived cardiac and brain microtissues (MTs), using a low adhesion cell suspension method, which display uniform size, shape and longevity with key tissue specific markers. Mitochondrial dysfunction and calcium homeostasis are commonly observed responses to toxic compounds and are implicated in neuro- and cardiotoxicity. Following 72h or 336h repeat dose exposure to a panel of in vivo neuro- or cardiotoxic categorized compounds, fluorescent probes were incorporated into each model. Multiplexed images were acquired using the confocal mode of an ArrayScan XTI HCS reader followed by ATP measurement. A 15x (brain MTs) or a 10x (cardiac MTs) Cmax cut off was applied. Following, 72h and 336h exposure, brain MTs correctly predicted 70% and 80% of the compound panel, respectively. With 72h and 336h exposure, cardiac MTs correctly predicted 75% and 88% of the compound panel, respectively. This study shows how using single organotypic iPSC derived 3D models per well and automated multiplexed confocal HCS can enhance the in vitro to in vivo translation of the potential for novel compounds to elicit toxic reactions.
113  Urine-sample-derived Human Induced Pluripotent Stem Cells As a Model to Study PCSK9-mediated Autosomal Dominant Hypercholesterolemia
Karim Si-Tayeb, INSERM
Abstract
PCSK9 is a critical modulator of cholesterol homeostasis. While PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypolipoproteinemia (FHBL). However, current cellular models to further decipher the role of PCSK9 mutations are limited. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSC) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine samples were used as a source of somatic cells in order to obtain hiPSC upon episomal vectors-mediated reprogramming. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, HLC-S127R (GOF) had a 71% decrease of LDL uptake, while HLC-R104C/V114A (LOF) displayed a 106% increase of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. In addition, pravastatin treatments led to an increase of LDL uptake of 2.19 ± 0.77 fold in HLC-S127R compared to 1.38 ± 0.49 fold in control HLCs in line with the good response of patients carrying the S127R mutation (mean LDL cholesterol reduction = 60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source for cells reprogramming and disease modeling.
114  HTRF Cell Signaling Platform Combined with iCell Hepatocytes: A Smart Solution to Study Insulin Resistance in Type 2 Diabetes
Brigitte Machard, Cisbio Bioassays
Abstract
For many years, researchers have utilized primary rodent cells or immortalized cell lines as in vitro models. In order to more closely resemble pathophysiology, Cellular Dynamics International (CDI) is developing innovative cellular models engineered from human induced pluripotent stem cells (iPSC), which can be differentiated into various terminal cell types, including iCell Hepatocytes. Activation of intracellular signal-transduction proteins is often analyzed by western blot, but this labor-intensive method often reveals a lack of accuracy and reproducibility. To overcome these limitations, Cisbio Bioassays is developing HTRF (homogeneous time-resolved fluorescence) cell-based sandwich immunoassays to analyze protein phosphorylation status and expression level. These homogeneous TR-FRET assays are quantitative, sensitive and HTS-compatible. Here, we demonstrate that iCell Hepatocytes in combination with HTRF phospho-/total protein assays, represent a straightforward and physiologically-relevant solution for the investigation of insulin resistance in T2D. Using both sets of tools, we dissected the insulin/AKT and AMPK/ACC pathways that are involved in glucose/fatty acid metabolism. The numerous stress pathways activated by pro-inflammatory cytokines, such as JNK, NFκB, p38 MAPK and STAT3, were also analyzed. Results obtained on iPSC-derived hepatocytes were compared with those obtained on the well-known immortalized HepG2 cell line.
201  Characterizing the Physiological Relevance of Functional Phenotypes of Human iPSC-derived Neuronal Cell Lines €“ A Tool for Quality Assurance in Cell-based Drug Discovery
Benjamin Bader, Neuroproof
Abstract
Generating neuronal cultures from human induced pluripotent stem cell-derived (hiPSC) is a promising approach to increase translation between in vitro tests and the in vivo situation in patients. Today, more and more protocols deliver neuronal cultures with spontaneous electrical activity, with different qualities however. Moreover, one of the most important concerns is their physiological relevance needed for disease modeling. Here, we aim to understand and compare the differences between multiple hiPSC neuronal cultures by phenotypically comparing them to a well-known reference: the robust electrical functional activity patterns from primary murine neuronal cell cultures recorded with micro-electrode arrays. Using multi-parametric analysis of MEA data, we generated a fingerprint for different primary cultures using hundreds of datasets and classified them correctly. Primary tissue cultures include frontal cortex, hippocampus, hypothalamus, midbrain/cortex and spinal cord. We cultured different lines of commercially available hiPSC neurons on MEAs and recorded their activity development for up to 4 weeks and also computed fingerprints at different developmental stages. We show that human neuronal cell lines exhibit specific phenotypic similarity profile when compared to the primary culture reference database, e.g. to hippocampus or midbrain or mixed similarities. Moreover, the similarity profiles can be changed by toxin or drug application. In conclusion, we provide a functional tool to characterize neuronal phenotypes from hiPSC neurons to either adapt their differentiation protocols or mixing neuron-specific cell lines to reach a more relevant phenotype, needed for disease-relevant in vitro modeling.
202  A Human 3D Neural Culture Model Utilising iPSCs from an Alzheimer Disease (AD) Patient to Study Neurodegenerative Diseases
Sandrine Willaime-Morawek, University of Southampton
Abstract
Organ specific research has advanced greatly through the use of biopsy tissues whereas the collection of live human brain tissue is relatively rare and live brain tissue from an AD patient almost unheard of. We can now obtain iPSCs, from the fibroblasts of AD patients, containing the exact genetic mutations that give rise to the disease. The next challenge is to create a neural tissue-like model with the genetic signatures of AD patients in which to study AD pathology. Using iPSCs from an AD patient containing the presenilin 1 mutation (L286V), we produced a robust 3D human neural culture system for the study of early onset AD and validated this model using immunofluorescence, western blot and electrophysiology. AD patient-derived neural progenitor cells were plated in Matrigel and self-organised into 3D networks by week 3. These cultures contained a heterogeneous cell population expressing markers for neurons and astrocytes and importantly AD-associated proteins tau and amyloid precursor protein/Abeta and were maintained in vitro for 18+ weeks. By 12 weeks these cultures contained maturing neurons capable of producing action potentials and repetitive firing. We have created 3D neural cultures derived from an AD patient to study the disease pathology in an isogenic robust and neuronally mature human model. We believe that this is a viable new in vitro model for the study of neurodegeneration, which can used as a powerful new tool in the fight against AD.
203  Pioneering iPS Cell Applications for Safety and Toxicity
Gesa Rascher-Eggstein, Nanion Technologies
Abstract
Induced pluripotent stem cells (iPSCs) emerged as the model of choice for cardiovascular risk assessment and for neuro- and hepatotoxicity screening in-vitro assays. Here, we report on optimized assays based on three different technologies. 1) We implemented a hybrid screening method that combines impedance (cell contractility) with MEA-like extracellular field potential (EFP) into routine screening of iPSC cardiomyocytes. Combined, this assay provides a non-invasive, label-free, high temporal resolution approach for screening iPSC derived cardiomyocytes. Furthermore, we present a new hepatotox screening assay on the impedance-based technology. 2) We developed miniaturized, modular patch clamp devices with full integration in automated robotic platforms, which enable parallel ion channel screening with a chip-based approach in the industry standard microtiter plate format. By drastically minimizing the total cell number required to execute the assays, we adopted our automated patch clamp instruments to iPSC-derived cells such as cardiomyocytes or neurons. 3) By using optogenetics combined with a MEA-based system, we present an in vitro assay with sensitive and multiple readouts for neurotox assays. The system allows, as well, to study iPSC-based neuronal disease models due to the high throughput, ultra-high resolution (millisecond events with microvolt amplitudes), high electrode count (allows population network activity measurements) and accuracy.
204  Generation of Human Naive Pluripotent Stem Cells through Reprograming: An In Vitro Model of Pluripotency Regulation
Stephanie Kilens, INSERM
Abstract
Recent advances in the human preimplantation development have highlighted transcriptomic and epigenetic differences between two pluripotent states: the naive pluripotent state in the preimplantation epiblast and the primed pluripotent state in the post implantation epiblast. Human pluripotent stem cells (hPSC) that were either derived from the embryo (hESC) or reprogrammed from somatic cells (hiPSC) are considered being in a primed pluripotent state. Therefore, there is a high need to model in vitro human naive pluripotency as a way to understand regulation of human pluripotency and X-inactivation process in female cells. We set up a protocol that allows reprogramming of human somatic cells into both human naive (hiNPSC) and primed (hiPSC) induced pluripotent stem cells. We were able to generate 8 transgene-free naive cell lines with their primed counterparts. We will present our preliminary results on the characterisation of our human naive cell lines in terms of transcriptomic and epigenetic features as well as metabolic activity and X-chromosome status for female cell lines.
206  Generation of Induced Pluripotent Stem Cells from Osteoarthritic Patients
Rocío Castro-Viñuelas, University of A Coruña
Abstract
Introduction: Induced pluripotent stem cells (iPSC) have emerged as a promising tool for modelling diseases. This approach allows researchers to further study pathological mechanisms and to test new drugs. Aim: To generate iPSC from human fibroblasts obtained from patients with different osteoarthritic (OA) joints in order to obtain iPSC-based OA models. Material and Methods: Fibroblasts were isolated from skin biopsies of one patient with OA knee, one with OA hand, and one healthy donor. These cells were histologically analyzed and karyotyped before reprogramming with Sendai virus. Cell lines obtained were clonally expanded and characterized. Results: Cells were isolated from the skin of OA patients and a healthy donor. Histological and immunohistochemical analyses showed that the 85-95% of cells in the culture were fibroblasts. These cells presented a normal karyotype. Three weeks after reprogramming, colonies presented embryonic stem cell-like morphology, besides positivity for alkaline phosphatase and pluripotent markers, such as Tra-1-81 and Nanog. Conclusions: Fibroblasts were successfully isolated and reprogrammed from OA patients. Further characterization analyses are currently being developed in order to establish the iPSC lines before using for OA modeling.
207  Compounds with Different Pharmacological Profiles Enhance the Neurite Outgrowth in Human iPSC-derived Neurons
Stéphanie Wagner, Neurofit
Abstract
There is a vast amount of evidence indicating that neurotrophic factors (neurotrophins) play a major role in the development, maintenance, and survival of neurons. Neurotrophic factors, which repair damaged neurons through stimulation of neurite outgrowth, may be important for the regeneration of the damaged neurons. The development of a new compound that could mimic the neurotrophin effect appears to be a good strategy for the development of new therapeutics in neurodegenerative diseases. In the present work, human iPSC-derived neurons (iCell Neurons) were used to assess the neuritogenic potential of different compounds with different pharmacological profiles. The results show that spontaneous neurite outgrowth occurred in human iPSC-derived neurons. The neurite outgrowth was time-dependent and reached a plateau around 14 days of culture. Brain-derived neurotrophic factor significantly enhanced the neurite outgrowth in iCell Neurons. Similar effect was observed with other types of neurotrophins such as fibroblast growth factor (FGF) and nerve growth factor (NGF). Furthermore, 2 drugs (donepezil and imipramine) of distinct mechanisms known for their neuritogenic potential showed a marked stimulation of neurite outgrowth in iCell Neurons. These results suggest that iCell Neurons respond to different mechanisms of neuritogenic agents and thus can be instrumental to screen neurotrophic compounds.
208  Using the Quasi Vivo System to Generate Gradients for the Differentiation, Maturation and Maintenance of iPSC Models
Stella Homer, Kirkstall
Abstract
Using the proprietary Quasi Vivo technology, cells cultured under optimum flow conditions are more metabolically competent than those cultured using traditional static techniques, and it appears that cells in interconnected chambers can create homeostatic conditions. Our currently available meso-scale Quasi Vivo technology can lead the way towards relevant and easy-to-use in vitro techniques for animal replacement. In a recently published paper, Rashidi et al. showed increased human-relevance of a hESC-derived hepatocyte model during drug challenge studies. In addition, during an Innovate UK funded grant with Locate Therapeutics (Nottingham, UK), we demonstrated the benefits of flow for primary human and rat MSCs over long time periods compared to static conditions. We have utilised the Quasi Vivo system in the development of many different models, including liver, cardiovascular, kidney and blood brain barrier. The Quasi Vivo system can be used to generate gradients of oxygen and nutrients, which could be harnessed to drive differentiation of iPSC cells towards specific cell types, and further to ensure that they mature into cells with mature phenotypes. We are currently looking for partners to develop new protocols for the differentiation, maturation and maintenance of iPSC-derived models.
209  Perturbation of ER-mitochondria Communication in Parkinson's Disease: A Study in Patient Fibroblasts and iPSC-derived Neurons
Zoi Erpapazoglou, Brain and Spine Institute (ICM)
Abstract
Parkinson's disease (PD) is a common movement disorder caused by progressive degeneration of the dopaminergic neurons of the substantia nigra pars compacta. Mutations in the PARK2 gene, coding for the cytosolic E3 ubiquitin protein ligase Parkin, are a common cause of autosomal recessive PD. Parkin plays a key role in mitochondrial quality control, and loss of its function affects mitochondrial biogenesis, bioenergetics, dynamics, transport and turnover. We investigated the effect of PARK2 mutations on the structural and functional properties of the endoplasmic reticulum (ER)-mitochondria interface, a subcellular compartment involved in Ca2+ exchanges between the two organelles. We demonstrated a significant increase in ER-mitochondria contacts in fibroblasts from PD patients with PARK2 mutations compared to cells from control individuals. Increased proximity between mitochondria and ER was accompanied by enhanced ER-to-mitochondria Ca2+ transfer and mitochondrial Ca2+ overload in fibroblasts with PARK2 mutations. Normal Ca2+ transients were restored in cells from a patient by PARK2 overexpression, or by partial down-regulation of mitofusin 2, a well-established substrate of Parkin at the ER-mitochondria interface. We found that cytosolic Ca2+ transients were also significantly different in induced pluripotent stem cells (iPSC)-derived neurons from a PARK2 patient compared to those from a control. Current work aims at generating isogenic control-patient iPSC pairs in order to further investigate the impact of PARK2 mutations on Ca2+ homeostasis and cell viability of in vitro differentiated dopaminergic neurons. These studies should provide new insight into how Parkin regulates mitochondrial physiology and how loss of its function contributes to the development of Parkinsonism.
210  Generation of Alzheimer's Disease (AD) Patients' Induced Pluripotent Stem Cells (iPSC)
Carole Crozet, INSERM
Abstract
Amyloid precursor protein (APP) and tau protein are two main molecular actors of neurodegenerative affections, which are of prime importance in human health (AD). In the present program, our objective is to investigate neuronal APP and tau protein processing and metabolism using biochemical tools, innovative detection methods and metabolic approach in two complementary situations: in vivo in patients, and in vitro in cell culture. The goal is to get a comprehensive proteomic view based on the parallel analysis of patient samples (CSF) and samples generated in neuronal differentiated human embryonic stem cell (hESC) and induced pluripotent stem cells (iPSC) derived from AD-patients fibroblasts. This project will offer the unique opportunity to combine state-to-the-art approaches to understand how the APP fragments and peptides are generated as well as the modifications of the tau protein in normal and pathological situation.
211  Functional Analysis of iPSC-derived Neurons inside a Research Core Facility Dedicated to Electrophysiology
Carine Dalle, Brain and Spine Institute (ICM)
Abstract
CELIS-EPHYS is a research core facility of the Brain and Spine Institute (ICM) that provides research services to academics and pharmaceutical industry requiring in vitro electrophysiology data. Among the different research projects supported by the facility, the project related to the stem cell field has increased exponentially these last years in correlation with the use and generation of induced pluripotent stem cell (iPSC)-generated cells by the ICM scientific community. The goals of CELIS-EPHYS is to facilitate the functional analysis of human iPSC-derived neurons by providing a complete set of electrophysiological data. We currently offer patch-clamp technology for (1) the functional characterization of different iPSC-derived cell types to determine their state of maturity (Gautier, Erpapazoglou et al., 2016) and (2) the study of the excitability of different patient-specific iPSC neurons. This functional analysis is essential to provide new, groundbreaking insights into disease mechanism and further to screen novel therapeutic compounds. In 2017, a new service will be launched to better meet investigator needs by offering the powerful research tool of the multi-electrode array (MEA) technology. CELIS-EPHYS is working in close collaboration with the core facility of ICM dedicated to iPSC technology (CELIS-iPS stem cell core facility).
212  iPS Cell Model for Hypertrophic Cardiomyopathy
Marisa Ojala, University of Tampere
Abstract
Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease, which affects the structure of the heart muscle tissue. The clinical symptoms include arrhythmias, progressive heart failure and even sudden cardiac death, but the mutation carrier can also be totally asymptomatic. To date, over 1400 mutations have been linked to HCM, mostly in genes encoding for sarcomeric proteins. However, the pathophysiological mechanisms of the disease are still largely unknown. Two founder mutations for HCM in Finland are located in myosin-binding protein C (MYBPC3-Gln1061X) and α-tropomyosin (TPM1-Asp175Asn) genes. We studied the properties of HCM cardiomyocytes (CMs) derived from patient-specific human induced pluripotent stem cells (hiPSCs) carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. Both types of HCM-CMs displayed pathological phenotype of HCM, but more importantly, we found major differences between CMs carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation in their cellular size, Ca2+ handling and electrophysiological properties, as well as in their gene expression profiles. These findings suggest that even though the clinical phenotypes of the patients carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation are similar the genetic background as well as the functional properties on the cellular level are different, indicating that the pathophysiological mechanisms behind the two mutations are divergent.
213  Characterization and Function of iPSC-derived Hepatocytes for Use in Toxicity
Mourad Ferhat, Promega France
Abstract
Hepatoxicity is a leading cause of drug withdrawal from the market, highlighting the fact that current preclinical models of toxicity are not universally predictive of drug effects in humans. The manifestations of hepatotoxicity are highly variable ranging from intrinsic toxic effects to the enzymatic production of toxic metabolites. Development of a more predictive model system that can elucidate the underlying mechanisms of hepatotoxicity early in the drug development process is critical to subverting unanticipated drug failure in the clinic. Cellular Dynamics International has developed human induced pluripotent stem cell (iPSC)-derived hepatocytes, iCell Hepatocytes, which exhibit biologically relevant functions necessary for hepatotoxicity studies. To demonstrate the functional utility of iCell Hepatocytes in elucidating mechanisms of drug-induced hepatotoxicity, a variety of functional endpoints were measured in response to known hepatotoxic compounds. Responses in iCell Hepatocytes were observed for all compounds at EC50s that were comparable to those seen with primary human hepatocytes. Mechanism-based toxicity was evaluated by analyzing cell metabolism, oxidative stress, and lysosomal phospholipase activity using multiple platforms, including luminescence-based assays (Promega). This multiple platform approach for functional analyses demonstrated how iCell Hepatocytes provide a biologically relevant human model system for studying drug-induced hepatotoxicity.
214  From Field Potential Waves to Ionic Current Knowledge by Leveraging Mathematical Modeling
Fabien Raphel, INRIA
Abstract
The use of microelectrode arrays (MEAs) and hiPSC-CMs allows high-throughput screening of NCEs or NBEs on human-derived cells. But the field potential (FP) signals are difficult to analyze. This work shows that an in silico approach can improve the practical use of FP signals. Method: We developed a new strategy consisting of a mathematical model which allow to analyze MEA and to derive ionic currents involved in the formation of the FP. The model is based on bidomain equations, coupled to a system representing the ionic channels. Various MEAs devices and ionic models (Paci., O'Hara-Rudy, MV) were considered. An inverse problem algorithm was proposed to identify ionic models parameters from measured FP. Results: In silico experiments provided new insights into some aspects of the FP difficult to address in vitro, like the potential variability due to the heterogeneity of cells. By extracting parameters from published FP data (depolarization/repolarization amplitude and FPD) for various drugs (e.g., Ivabradine, Moxifloxacin, Diltiazem, SEA0400), the proposed identification algorithm provided concentration-response curves for potassium, sodium and calcium channels. IC50 determined by simulation were in good agreement with literature values for Ivabradine and Moxifloxacin. Conclusion: The proposed in silico-in vitro signal analysis method may be a powerful addition to current CiPA approaches since it can provide ionic channel profiling from in vitro MEA data.
215  MAST FAST €“ Rapid Generation of Human and Mouse Mast Cells from ESCs/iPSCs
Mari-Liis Kauts, University of Edinburgh, Queens Medical Research Institute
Abstract
Mast cells are the major effectors of allergic responses such as hayfever, food and skin allergies, and asthma. Other medical conditions associated with mast cell disorders include migraine headache pain, anaphylaxsis, mastocytosis, autism, chronic fatigue syndrome, and neuroinflammation or pain in endometriosis. Current knowledge of the development and differentiation of the mast cell lineage in the context of health and disease is limited. Drug discoveries and new treatment strategies to counteract the damaging effects of mast cell disorders are hampered due to the difficulty in obtaining sufficient numbers of these cells for research. Here we describe a novel method for the robust and rapid production of highly proliferating immature and mature mast cells. It utilizes mouse embryonic stem cells (ESCs) engineered with a unique reporter gene (Gata2Venus) that allows for high enrichment of the mast cell precursors. Our data show that this protocol can yield 3-13 fold more mouse mast cells with only 18 days of culture as compared with previous studies that start from 4-5 weeks. The novel protocol can be effectively applied to human pluripotent stem cells (ESCs and induced pluripotent stem cells (iPSCs)). Our preliminary data indicate that phenotypic mast cells are generated after only 12 days of Gata2Venus ESC/iPSC differentiation cultures. This is an important improvement in the field as current protocols generate human mast cells from ESCs/iPSCs starting from 10-15 weeks of culture and yield low numbers. Furthermore, our protocol can be effectively applied to human iPSCs generated from patients with mast cell-associated hematological disorders/allergies/inflammation. This allows to test patient-specific treatment strategies and to screen for putative drug candidates opening the field for personalized medicine.
216 iPSC Generation with a Non-integrative and FDA Approved System
Beatriz Fernandez Munoz, LARCEL
Abstract
Our aim is to develop a protocol for iPSC generation more amenable for a clinical use. First we compared the efficacy of fibroblasts isolation from different areas of the body using both mechanical and enzymatic protocols. Two skin biopsies from different donor areas were processed by either the collagenase technique or "explants technique", comparing their efficiency in isolating fibroblasts in terms of time and number of fibroblasts obtained. We found that the "explant technique" is a more efficient approach to obtain fibroblasts under our experimental conditions. The next step has been the optimization of the reprogramming method by an electroporation system approved by the FDA (MaxCyte GT system). We electroporated fibroblasts from 5 different donors with several electroporation programs. We then selected the best program with these data and we used this program to generate iPSC lines from 2 adult donors. We obtained an average of 20 colonies for each donor (efficiency of 0.001%). iPSC lines generated from each donor were evaluated for pluripotency by different tests: expression of pluripotency markers, karyotyping, lost of episomal vectors and in vitro differentiation test. We found that 100% of the lines were positives for at least one of the pluripotency markers. Here we demonstrate that generation of iPSC with this FDA approved and closed electroporation system is possible and has a similar efficiency to other protocols using episomal vectors.

Details

Date:
October 11, 2016
Website:
https://iforum.community/iforum-europe/

Venue

Institut du Cerveau et de la Moelle Epinière
47 Boulevard Hôpital
Paris, 75013 France