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Improving models of microglia: the development of physiologically functional human iPSC-derived microglia
Microglia are commonly described as the immune cells of the brain. Under physiological conditions, they are vigilant guard keepers of their microenvironment, seeking out invading pathogens and clearing up cell debris, apoptotic cells, and misfolded proteins by migrating, phagocytosing, and producing cytokines and neurotrophins; all to maintain a homeostatic balance in the CNS.
Axol’s method for generating hiPSC-derived microglia mimics the in vivo pathway of development for brain resident macrophages and produces high yields of microglia that are homogenous and representative of primary human microglia in vitro.
Human induced pluripotent stem cell-derived sensory neurons (hiPSC-sensory neurons) offer a physiologically relevant in vitro human model of pain perception. The dorsal root ganglion (DRG) is the collection of sensory neuron cell bodies which project axons into the peripheral nervous system. These sensory neurons express key and unique nociceptors which are implicated in chronic pain conditions.
Here we present data on the characterization of hiPSC-sensory neurons assessing the expression and function of the DRG-specific voltage-gated sodium channels (Nav1.7, Nav1.8 and Nav1.9) and transient receptor potential (TRP) ion channels, TRPV1, TRPA1 and TRPM8. Functional responses were evaluated against typical pain inducing molecules and chemotherapy drugs.
This data provides an in-depth characterization of Axol’s human iPSC-sensory neurons demonstrating a viable human cell-culture model for pioneering research and drug discovery on both nociceptive and neuropathic pain disorders.
Electrophysiological pain responses in cultured human iPSC-derived sensory neurons using high-throughput multi-electrode array system
The purpose of this study was to evaluate the physiological responses against typical pain-related molecules, temperature change and chemotherapeutic drugs Axol’s Human iPSC-derived Sensory Neurons using high-throughput multi-electrode array (MEA) system.
Dorsal root ganglion (DRG) sensory neurons are pain-related neurons and have a variety of sensory receptors that are activated by chemical, thermal, and mechanical stimuli. Establishment of pharmacological assay in pain research and drug screening is important issue. In addition, human induced pluripotent stem cell (hiPSC)-derived sensory neurons may be effectively used for drug discovery and toxicity testing.
Electrophysiological responses of cultured human iPSC-derived neuronal networks to convulsants or anti-convulsant drugs
The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating drug toxicity. Epileptiform activity is one of phenomena in neuronal toxicology. To evaluate the dynamics of epileptiform activities and the effect of anti-convulsant drug in cultured hiPSC-derived neurons, we used the high-throughput multielectrode array (MEA) system, where we simultaneously record extracellular potentials for 16 channels per well across 24-well plates.
We examined chemically evoked epileptiform activity. Epileptiform activityies were induced by 4-Aminopyridine (4-AP), pilocarpine, chlorpromazine, and pentylenetetrazole (PTZ) . The number of synchronized burst firings were increased in a concentration dependent manner at 4-AP, Pilocarpine, and Chlorpromazine administration. On the other hand, the duration and spikes in a synchronized burst were increased at PTZ administration. Phenytoin used in anti-convulsant drug suppressed electrophysiological activities. From these results, we suggest that the electrophysiological assay in cultured human iPSC-derived neuron using MEA system has the potential to investigate the neuronal toxicity in drug screening.
Dorsal root ganglion (DRG) sensory neurons are pain-related neurons and have a variety of sensory receptors that are activated by chemical, thermal, and mechanical stimuli. Establishment of pharmacological assay in pain research and drug screening is important issue. In addition, human induced pluripotent stem cell (hiPSC)-derived sensory neurons may be effectively used for drug discovery and toxicity testing. The purpose of this study was to evaluate the physiological responses against typical pain-related molecules, temperature change and anticancer drug in cultured sensory neurons using high-throughpupt multi-electrode array (MEA) system.
Next-generation neurological disease models: isogenic tools for investigating frontotemporal dementia, Alzheimer's and Parkinson's diseases
Alzheimer’s and Parkinson’s diseases are incurable and debilitating neurodegenerative conditions with strong links to age. Today Alzheimer’s disease alone accounts for 60-70% of dementia cases and Parkinson’s disease is expected to affect ~2% of individuals over 65 years. The human and economic impact of these conditions is expected to increase significantly with the increasingly aging global populations unless new therapeutic strategies can be developed. Currently, there are a lack of human cell-based models available in which to carry out such studies and further investigation is needed in order to elucidate disease mechanisms and determine the efficacy of novel drug compounds. iPSC-derived neural stem cells (NSCs) offer a virtually unlimited source of physiologically relevant isogenic lines for use in both disease modelling and drug discovery.
Combining the powerful tools of iPSC genome editing using CRISPR-Cas9 and directed differentiation, we have generated patient relevant NSC disease models carrying Alzheimer’s disease-associated microtubule-associated protein tau (MAPT) mutations, R406W, P301L and V337M and Parkinson’s disease-associated leucine-rich repeat kinase 2 (LRRK2) mutation, G2019S in both a heterozygous and homozygous manner. These clinically identified missense mutations in MAPT are thought to reduce the ability of tau to promote microtubule assembly and may contribute to neuronal death in Alzheimer’s disease. LRRK2 is thought to contribute to Parkinson’s disease via pathological mechanisms involving tau, oxidative stress, α-synuclein, and mitochondrial-synaptic-dysfunction.
Genome-edited iPSC lines were genotyped, karyotyped and subsequently differentiated using fully-defined, xeno-free neural induction conditions (Shi et al et al., 2012). Once the cells formed polarized neural tube-like rosette structures in monolayer culture, immunocytochemistry confirmed the expression of typical cerebral cortical NSC markers namely, PAX6 and FOXG1.
These genetically defined, functionally validated human iPSC-derived NSCs provide a renewable resource of disease- and biologically-relevant cells. These cells, carrying relevant mutations, offer a stable platform on which novel therapeutic agents can be screened and validated. Furthermore, these cells enable a direct comparison of the variant effect on cellular phenotype between isogenic lines cells and may therefore provide further insight into the pathology of these diseases.
Functional phenotypic characterization of iPSC-neurons from Alzheimer’s disease patients carrying PS-1 mutation in drug screening and disease modeling
Adult cells from human individuals carrying disease-associated gene mutations can be reprogrammed into induced pluripotent stem cells (iPSCs) and can then be differentiated into a variety of cell types including human neural stem cells (hNSCs) and cerebral cortical neurons (hCCNs). Our aim was to phenotypically investigate patient iPSC-derived neurons carrying the presenilin-1 (PS-1) mutation (supplied by Axol Bioscience) and to compare them with cells from healthy controls (supplied by Axol Bioscience).
Transcriptome analysis revealed an up-regulation in the expression of neuronal genes and a decrease in pluripotency markers in hCCNs. Immunocytochemistry showed the appropriate neural cell morphology in hCCNs, with both cell types expressing markers typically associated with the corresponding developmental stage. Whole patch clamp and multi-electrode arrays (MEAs) successfully established electrical activity in these cells.
We differentiated these neural progenitor cells into spontaneously active neuronal networks using a xeno-free differentiation protocol and recorded spontaneous activity during neuronal differentiation using micro-electrode array (MEAs). Multi-parametric phenotypic analysis was used to identify specific differences of functional activity patterns during development into mature neuronal networks within 4-5 weeks. Moreover, we investigated the effects of neurotoxins on mutant and control neurons. We have identified a range of characteristics in the patient-derived and control- derived iPSC-neurons that establishes them as an ideal tool for use in numerous applications such as disease modeling, drug screening and toxicology and other assays.
Adult cells can be reprogrammed by defined factors, OCT3/4, KLF4, SOX2 and c-MYC, to generate induced pluripotent stem cells (iPSCs). These can then be differentiated into a variety of cell types including human neural stem cells (hNSCs) and cerebral cortical neurons (hCCNs). Reprogramming and differentiation can be carried out on cells from healthy donors and patients suffering from disease and could potentially be used as a model in the study of human neuronal development and disease. In order to determine the suitability of these iPSC-derived cells for neurobiological research, we conducted a series of e xperimental procedures to examine the functional characteristics of these cells and their progeny in vitro. Transcriptome analysis revealed an up-regulation in the expression of neuronal genes and a decrease in pluripotency markers in hNSCS. Immunocytochemistry showed the appropriate neural cell morphology in hNSCs and hCCNs, with both cell types expressing markers typically associated with the corresponding developmental stage. When cultured in 3D using a collagen matrix, both cell types formed common neural structures. hNSCs yielded the highest neurite length and branch point values when compared to a variety of other neural cell types. Whole patch clamp and multi-electrode arrays (MEAs) successfully established electrical activity in these cells. We have identified a range of characteristics in the hNSCs and hCCNs that establishes them as an ideal tool for use in numerous applications such as disease modelling, drug screening and toxicology and other assays.
Modelling neurological disease: in vitro gene editing and iPSC differentiation combine to create powerful new tools
Neurodegenerative diseases, such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and other age-related dementias are incurable and debilitating conditions, with Alzheimer’s disease alone accounting for ~60-70% of cases. With an increasingly ageing global population, the economic as well as human impact of these conditions is expected to increase unless novel therapeutics and care strategies can be developed. Induced pluripotent stem cells (iPSCs) and gene-editing technology, offers unprecedented biomedical potential for disease modelling, high-throughput drug screening and development of therapeutic strategies for such diseases.
We have generated stable human iPSC lines from normal human dermal fibroblasts and patient derived fibroblasts (e.g. Huntingdon’s and Alzheimer’s diseases). The fibroblasts were reprogrammed using a non-integrating episomal method coding for Yamanaka factors (license agreement with iPS Academia Japan) and then differentiated into neuronal stem cells (NSCs) and cortical neurons to provide a complete modelling solution in a dish. The iPSC lines derived from normal human dermal fibroblasts were stable with all the hallmarks of pluripotency and a normal karyotype for over 13 passages. These could be cultured as single cells, an essential prerequisite for efficient genome editing. Using the CRISPR-Cas9 genome–editing technology, we generated patient relevant disease models carrying microtubule-associated protein Tau (MAPT) mutations. Tau protein is normally associated with microtubules and is involved in their assembly and stabilization. In turn, microtubules are critical for cellular function, especially for neurons to facilitate the growth and integrity of axons and dendrites and transport between the cell body and distant dendrites. Clinically identified missense mutations reduce the ability of Tau to promote microtubule assembly, resulting in neuronal cell death and subsequent disease phenotype.
These renewable and biologically relevant resources will further enable investigation of the mechanisms of disease progression, with additional models relevant to Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease and epilepsy being generated to aid in the identification of novel drug discovery targets.
Long-term potentiation (LTP) and long-term potentiation depression (LTD) in neuronal networks has been analyzed using in vitro and in vivo techniques in simple animals to understand learning, memory, and development in brain function. Human induced pluripotent stem cell (hiPSC)- derived neurons may be effectively used for understanding the plasticity mechanism in human neuronal networks, thereby elucidating disease mechanisms and drug discoveries. In this study, we attempted the induction of LTP and LTD phenomena in a cultured hiPSC-derived cerebral cortical neuronal network using multi-electrode array (MEA) systems. High-frequency stimulation (HFS) produced a potentiated and depressed transmission in a neuronal circuit for 1 h in the evoked responses by test stimulus. The cross-correlation of responses revealed that spike patterns with specific timing were generated during LTP induction and disappeared during LTD induction and that the hiPSC-derived cortical neuronal network has the potential to repeatedly express the spike pattern with a precise timing change within 0.5 ms. We also detected the phenomenon for late-phase LTP (L-LTP) like plasticity and the effects for synchronized burst firing (SBF) in spontaneous firings by HFS. In conclusion, we detected the LTP and LTD phenomena in a hiPSC-derived neuronal network as the change of spike pattern. The studies of plasticity using hiPSC-derived neurons and a MEA system may be beneficial for clarifying the functions of human neuronal circuits and for applying to drug screening.