SfN’s 49th annual meeting is the premier venue for neuroscientists to present emerging science, learn from experts, forge collaborations with peers, explore new tools and technologies, and advance careers. Neuroscience 2019 will take place October 19-23 at McCormick Place in Chicago. Join 30,000 colleagues from more than 70 countries at the world’s largest marketplace of ideas and tools for global neuroscience.
Come visit us at Booth # 354 and learn about the latest from FCDI including new iCell® Microglia disease models including:
- SNCA (A53T) for Parkinson’s Disease
- TREM 2 mutants for Alzheimer’s Disease
- MECP2 for Rett Syndrome and other neurodevelopmental disorders
Meet with us at Neuroscience 2019
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Title: Modelling Neuroinflammation using iPSC-derived Engineered Microglia
Time & Location: Mon 10/21 poster #373.20 /C31
Authors: Beatriz Freitas, Michael McLachlan, Sarah J Dickerson, Christie A Munn, Sarah A Burton, Abbey Musinsky, Madelyn Goedland, Anne Strouse, Deepika Rajesh, Simon Hilcove and Eugenia Jones – FUJIFILM Cellular Dynamics, Inc. Madison WI
Abstract: Neurodegenerative and neurodevelopmental disorders can display aspects of neuroinflammation with microglial cells as crucial players during this detrimental stage. Microglia, the resident immune cells of the central nervous system, are a necessary cell type for neuronal homeostasis and are also responsible for synaptic pruning and brain development. Human iPSC-derived microglia can serve as an authentic preclinical tool for understanding the pathobiology of neurodegenerative and neurodevelopmental diseases. The present study involves the generation and characterization of genome engineered iPSC-derived iCell® Microglia to facilitate disease modeling of neurodevelopmental (Rett Syndrome) and neurodegenerative disorders (Alzheimer’s and Parkinson’s Disease). iCell Microglia generated from both the engineered and non-engineered iPSC clones offer unique isogenic pairs for research applications.
Microglia were derived by first by differentiating iPSCs into purified hematopoietic progenitor cells (HPCs), which were then further differentiated into microglia using technology developed by the Blurton-Jones laboratory (Abud et al. Neuron 2017) for which FujiFilm Cellular Dynamics Inc. has an exclusive license from the University of California-Irvine. iCell Microglia were highly characterized by assessing: (i) morphology; (ii) protein expression of TREM2, P2RY12, CX3CR1, IBA1, CD33 and CD45 by flow cytometry; (iii) phagocytic function using pHrodo-labelled BioParticles and aggregated amyloid beta (iv) quantification of neuroinflammatory molecules by multiplex Luminex; (v) measurement of soluble TREM by ELISA; and finally (vi) RNAseq analysis. Cryopreserved microglia retained purity and function comparable to pre-cryopreserved end-stage microglia. These results identified critical differences between wild type (WT) and engineered microglia in survival, phagocytosis kinetics, and expression levels of molecules involved in neural inflammation. Thus,these data demonstrate that iPSC-derived isogenic WT and engineered microglia could serve as a powerful tool to gain insight into various physiological and pathological conditions associated with neurological disorders and serve as a reliable in vitro disease model for complex neuronal diseases.
Title: Parkinson’s Disease-related Phenotype Characterization of A53T Alpha-synuclein iPSC-derived Dopaminergic Cultures
Time & Location: Sat 10/19 poster #043.01/C75
Authors: T. Ferraro, R. Remelli, E. Bianchini, E. Torchio, A. Toti, C. Griffante, M. Corse; In Vitro Pharmacol., Aptuit Srl, an Evotec Co., Verona, Italy
Introduction: Parkinson disease (PD) is a progressive neurological disease caused by selective loss of dopaminergic (DA) neurons in the substantia nigra. Although the majority of PD cases are sporadic, familial PD mutations provide a valuable tool for understanding and modelling basic pathophysiological mechanisms. We used MyCell® DopaNeurons A53T carrying the A53T mutation in the SNCA gene and healthy isogenic control iCell® DopaNeurons (FujiFilm) to investigate disease-relevant phenotypes including alpha synuclein (αSyn) accumulation, calcium dysregulation and mitochondrial dysfunction. Methods and Results: Cell culture and neurons differentiation was carried out in 384-well plate format. High content imaging studies revealed that more than 70% of viable neurons were tyrosine hydroxylase-positive at day 14 post-seeding in both A53T and control DA cultures, with comparable viability up to day 28. Since A53T mutation is reported to induce αSyn protein accumulation and aggregation, we sought to determine whether the A53T culture recapitulates this phenotype using Meso Scale Discovery®, a highly sensitive and quantitative technology. Results revealed a time-dependent selective accumulation of αSyn in A53T cultures, with a maximal difference between the two cultures detected at day 28 (1.61 ±0.18 folds, three independent cultures). Furthermore, spontaneous calcium oscillations were studied by applying FLIPR® technology. A different pattern of calcium oscillations was observed between A53T and control mature cultures. A53T neurons displayed 1.5-fold higher peak frequency and average peak amplitude compared to controls, suggesting a dysregulation of intracellular calcium homeostasis, being associated with mitochondrial oxidative stress. Mitochondrial membrane potential (MMP) was used as a read-out of possible mitochondrial dysfunction. A high content imaging assay combining cell viability and MMP quantification showed a 10% to 20% higher depolarization of mitochondria in A53T cultures compared to controls at different time-points (day 28-35).
Conclusions: Key cellular features linked to PD pathogenesis were identified in A53T iPSC-derived DA cultures. Based on this phenotypic characterization, an array of different assays was developed and will provide a valuable tool for the identification of neuroprotective compounds in PD drug discovery programs.
Title: Engineering 3D Neural Tissues for Drug Screening
Time & Location: Wed 10/23 poster #704.01 / CC61
Authors: A. N. Vo, S. Kundu, M. J. Song, M. Ferrer, M. E. Boutin, Natl. Ctr. for Advancing Translational Sci., Rockville, MD
Abstract: In 2017, the opioid crisis in the U.S. was declared a public health emergency. To help combat this crisis, the 3D Tissue Bioprinting Laboratory at NCATS is supporting the NIH Helping End Addiction Long-Term (HEAL) Initiative by developing morphologically- and physiologically-relevant human 3D neural tissue models for the discovery of new treatments for pain and opioid addiction and overdose. Predictive in vitro 3D neural tissue model systems must contain functional mixtures of neurons and glial cells of the brain and mimic the native brain microenvironment. Our group is developing 3D biofabricated neural tissue models using human induced pluripotent stem cell (iPSC)-derived neuronal and astrocyte cells with macromolecular crowding (MMC) agents to induce extracellular matrix (ECM) production and deposition. Initial studies were performed in 2D monocultures to identify media which supports spontaneous neuronal activity in human iPSC-derived dopaminergic neurons and determine MMC agents which induce deposition of endogenous matrix by astrocytes. Among the tested media, neurons seeded in iCell Complete Maintenance Medium and transitioned to BrainPhys complete media on 2 days in vitro were found to have the most consistent spontaneous firing over a 28-day period. MMC optimization studies show that addition of Ficoll 70/400 crowding with ascorbic acid in astrocyte monoculture increased secretion and deposition of ECM proteins collagen I, collagen IV, and laminin. Ongoing studies include testing the optimal medium with MMC for co-culture of neurons and astrocytes in 3D and optimizing the hydrogel components for the support of the 3D co-cultures. Future studies aim to utilize this 3D culture models to develop in vivo-relevant assays for HEAL-related experiments.
Title: A Neuronal Dynamic Clamp System for Improving Action Potential Recording in Human Stem Cell Derived Neurons
Time & Location: Mon 10/21 poster #431.04 / DD9
Authors:M. W. NOWAK1, B. K. PANAMA2,1, B. FRANKS1, L. KORBEL1, R. L. RASMUSSON2,1, G. C. L. BETT2,1
- Cytocybernetics Inc., North Tonawanda, NY;
- SUNY Buffalo, Buffalo, NY
Abstract: Human stem-cell derived (hiPSC) neurons are a model system for studying the electrophysiological properties of native neurons. A significant limitation of the neuronal hiPSCs is the decreased/lack of expression of background K+ currents (e.g. TASK, TWIK, TREK) resulting in depolarized resting membrane potentials (RMPs) and the inability to evoke stable action potentials (APs). We have overcome this limitation by using a neuronal dynamic clamp system to electronically express in real-time tunable K+ current-voltage relationships to hyperpolarize the RMP. iCell hiPSC GABANeurons (Fujifilm Cellular Dynamics, WI) were cultured according to the manufacturer’s instructions and used 7-27 days after plating. Standard electrophysiology voltage clamp with the whole-cell ruptured configuration was used, and APs were triggered by stimulating with 0.5-1.0 nA pulses for 0.3-1.5 ms at 0.5 Hz. Background K+ currents were electronically expressed using a real-time dynamic clamp system (Cytocybernetics, NY). The resting membrane potentials (RMP) of the neuronal hiPSCs were highly depolarized (31±1 mV, n=72) with distorted evoked APs (Amp. = 60±3 mV, after-hyperpolarization (AHP) at -45±2 mV). We tuned the RMP to -65 mV via injection of a constant current (Iconst) and the electronic expression of a background outwardly rectifying K+ current (IGHK). Injection of a constant current resulted in unstable RMPs (σ=2; n=8) while electronic expression of IGHK had a more stable RMP (σ=0.7 mV, n=10). There were differences in AP morphology. With expression of IGHK the amplitude showed a decrease as compared to constant current injection (IGHK: 112±4 mV, Iconst=123±4 mV, n=8, p<0.05). To examine the effects of background conductance on excitability we extended the electronic expression of IGHK to allow tuning of RMP to hyperpolarized potentials (-55 to -65 mV) resulting in the recording of evoked APs with a characteristic after-hyperpolarization (for RMP= 55 mV: Amp.= 95±4 mV, AHP at 62±1 mV, n=25). In a subset of hiPSC neurons, we also observed RMP-dependent spontaneous firing. Taken together, we have shown that electronic addition of background K+ currents during electrophysiological recording in hiPSC neurons allows the tuning of RMPs to physiological levels improving the measurement of functional responses (action potential morphology, phasic firing, post synaptic summation, after-hyperpolarization and long-term potentiation).