Pharmacological evaluation in human iPSC-derived cortical and sensory neurons using high-throughput MEA system
Human iPSC-derived neurons are expected as a new toxicological evaluation assay to replace animal experiments in preclinical studies, improving the risk of accuracy.
The most sensitive microelectrode array system for in vitro extracellular electrophysiology
MEAs have been adapted to drug discovery assays including cell-to-cell interaction, learning and memory, and network properties of neurons. High sensitivity consists key factor of their performance; it results in better signal to noise ratio leading to more reliable detection of action porentials. Michael Trujillo, Product Manager at Alpha Med Scientific, explains how low noise in engineered into MED64 systems.
Relevance of iPS cells in cell based screening and drug development
Drug development requires highly validated, reproducible, consistent and predictive models in order to quickly validate key targets and assess toxicity in pre-clinical assays.
High quality human iPSC cells can help to that direction and Nick Clare analyses further in his presentation at ELRIG Drug Discovery 2017 how that can be achieved.
Induced pluripotent stem cell-derived endothelial colony forming cells offer a robust and physiologically relevant research tool
Human iPSC-Derived Endothelial Colony Forming Cells (ECFCs) (Axol Bioscience) are highly expandable and show comparable expression and functionality to primary cells, providing a robust and physiologically relevant tool for use in numerous applications. ECFCs are rare circulating endothelial cells that display a hierarchy of clonal proliferative potential and possess in vivo vessel-forming ability upon implantation. In numerous animal models of disease, human ECFCs have demonstrated the capacity to promote revascularization and reperfusion to injured vascular beds via direct integration and/or through paracrine effects. Human umbilical cord blood is enriched in circulating ECFCs compared to adult peripheral blood. We have recently reported that ECFCs displaying properties similar to cord blood can be obtained from human ESCs and iPSCs. Using defined serum-free culture medium and sequential addition of specific growth factors, we’ve identified ECFC precursors within 12 days of iPSC differentiation. These iPSC-derived ECFCs display clonogenic proliferative potential and in vivo vessel forming ability similar to cord blood ECFCs and promote vascular repair and regeneration in multiple animal models of human disease.
An overview of Axol Bioscience
Axol Bioscience specialize in the supply of human cell culture systems and custom services for disease modeling and drug discovery. Our expertise includes induced pluripotent stem cell (iPSC) generation, CRISPR-Cas9 gene editing, iPSC differentiation and custom cell and tissue sourcing.
We offer a variety of functionally validated human primary cells and human iPSC-derived cells from both healthy and patient donors alongside culture media and reagents that are tailored to support the optimal growth and maturation of our cells. This enables you to carry out your project in a physiologically relevant system that is not only reliable and easy to use but produces consistent results for robust replication studies.
Our team is passionate about great science, delivering superb customer service and support and innovating future products to help customers advance their research faster.
At Axol, innovation, quality and customer service are key.
Frontotemporal dementia and Parkinson's Disease cellular models: In vitro characterization of pathological phenotypes in gene-edited iPSC-derived neurons with MAPT and LRRK2 mutations
Mutations in MAPT and LRRK2 are reported to play a causative role in familial forms of frontotemporal dementia and Parkinson’s disease respectively. Human cell-based models in which to investigate the effect of these mutations on cellular functions such as microtubule assembly, oxidative stress, mitochondrial- and synaptic-functions, all of which could contribute to the onset of neurodegenerative conditions, are necessary to understand the disease mechanisms. Genome-edited human iPSC-derived neural stem cells (NSCs) offer a virtually unlimited source of physiologically relevant isogenic cell lines for use in disease modelling and drug discovery. Combining iPSC genome editing using CRISPR-Cas9 and directed differentiation, we have generated patient-relevant NSC disease models (axolGEMs) carrying heterozygous and homozygous combinations of missense mutations R406W, P301L and V337M in MAPT and G2019S in LRRK2. Genotype, karyotype, SNP frequency and copy number were assessed in genome-edited iPSC lines. Differentiated cells formed polarized neural tube-like rosettes in a monolayer culture. Immunocytochemistry confirmed the expression of typical NSC (PAX6, FOXG1, nestin, Ki67 and NCAD) and cerebral cortical neuronal markers (TUJ1, TBR1, MAP2 and CTIP). These cells enable a direct comparison of the variant effect on cellular phenotype between isogenic lines and offer a stable platform for drug screening and validation.
Abnormal phosphorylation of amyloid precursor protein tyrosine residues alters the APP trafficking in neurons from Alzheimer's Disease affected patients
Alzheimer’s Disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-β (Aβ) APP peptide byproduct. Understanding how APP is targeted and trafficked to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aβ are thus the key for the development of new therapies. We previously developed a mouse model showing that the Tyrosine (Tyr)682, in the C-terminus of APP, is essential for its binding to the coating protein clathrin and to the clathrin adaptor protein AP2 and for APP trafficking and sorting in neurons. In the present study, we investigated whether the Tyr682 residue of APP influences APP trafficking and sorting in neurons from differentiated neural stem cells (NSCs) of patients with AD carrying mutations on the PS1 gene (L286V; A246E; M146L), and on cortical tissues and fibroblasts from Göttingen minipigs engineered to develop AD (M146I mutant minipigs). Our results show that Tyrosine phosphorylation controls the APP binding to clathrin and AP2 in neurons carrying PS1 mutation and influences APP trafficking and sorting. Overall, our results provide a basis for the development of potential new therapies for AD.
Modeling FTDP-17 linked tauopathies and Alzheimer's disease with human iPSC
Dr An Verheyen, Janssen R&D Discovery Services presented her work at ISSCR Basel, 2017. Dr Verheyen used human iPSC-derived neural cells generated using our custom differentiation service to model tauopathies linked to frontotemporal dementia with parkinsonism-17 and Alzheimer's disease.
Electrophysiological maturation and pharmacological responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture
Ikuro Suzuki, Associate Professor at Tohoku Institute of Technology, Japan who recently published his work with Axol Human iPSC-Derived Cerebral Cortical Neurons in Nature’s Scientific Reports (Odawara et al., 2016), presented our Innovation Showcase in collaboration with Alpha MED Scientific.
Prof Suzuki discussed the following results from his research:
- Morphology in long-term culture
- Development of spontaneous burst firings
- Pharmacological properties
- Induction of epileptiform activity & effects of anti-epilepsy drugs
- Induction of long-term potentiation and depression
Human iPSC-derived cardiomyocytes: A comparison with primary cells and applications in standard and 3D culture models
Ventricular cardiac muscle cells, the cardiomyocytes, not to be confused with smooth muscle myocytes of the arterial wall or myoblasts of skeletal muscle, are the working muscle cells of the heart that relentlessly maintain the body’s circulation during a lifetime. Their high metabolic activity and low ischemic tolerance, sensitivity to changes in extracellular calcium, refractoriness to DNA transfection and limited lifespan in culture, notably without proliferation, make these cells a demanding in vitro model system. This issue is compounded by the absence of immortalized cell lines with characteristics comparable to primary human cardiomyocytes. The technology of reprograming somatic human cells into induced- pluripotent stem cells (hiPSC), which theoretically allows the production of cardiomyocytes and other cardiovascular cell types in unlimited amounts, has become popular as an alternative to primary rodent cells for disease modeling and toxicology. Furthermore, in vitro studies are now possible with hiPSC-derived cardiomyocytes from patients with specific disease-causing genotypes and genetic backgrounds, sometimes with access to their entire medical history.
Applications of hiPSC include general toxicology, drug efficacy and safety testing, cell physiology, disease modeling, 3D culture models and tissue engineering, and basic science. The only currently remaining concern with these cells is their comparably immature developmental state, showing features of the fetal or neonatal heart. In addition, there is heterogeneity in the cell population regarding expression patterns of different chambers of the heart or of the electrical conduction system. Also, some cellular features are less well formed such as T-tubules and the expression levels of connexins, depending on the properties of the original hiPSC-cell lines in comparison with the fully differentiated cells of the adult human heart. Several academic groups and companies are currently developing protocols and strategies to improve maturation and purity of hiPSC-derived cardiomyocytes. Our lab, at the University Hospital Bern, has been active in the field of cardio-toxicity for some years now, first with isolated cardiomyocytes from adult rats, using human samples from cardiac surgery, and now we are using hiPSC-derived cardiomyocytes from several commercial sources. As we are interested in the mechanisms of cancer therapy-associated cardiotoxicity, and want to study this in cardiomyocytes, we always endeavor to use the most relevant in vitro culture systems. Therefore, we have recently started to develop a 3D-culture model using hiPSC-derived cardiomyocytes and have tested this system in comparison with mature primary cells.
Modelling Alzheimer's Disease
Dr Alfredo Cabrera, Janssen Pharmaceutica, presented a robust, scalable and disease relevant model of tau aggregation using induced pluripotent stem cell (iPSC)-derived cortical neurons that can be applied to drug discovery programs in neurodegeneration. The resulting assay is highly reproducible across users and works in different commercially available iPSC-lines, providing a reliable tool for better understanding TAU pathophysiology and the identification of novel treatments against Alzheimer’s disease.
Dr Cabrera covers the following in this tutorial:
- iPSC and related initiatives in Europe: European Bank for Induced Pluripotent Stem Cells (EBiSC)
- Introduction to Alzheimer’s disease & tau aggregation
- Modelling tau aggregation using iPSCs
The use of iPSC-derived cells and primary cells as in vitro models for toxicity screening
Toxicologists have access to a range of iPSC-derived cell types, including cardiomyocytes, hepatocytes and renal cells, used in toxicity screening. We discuss how these models are accurate and representative cell models, and how they can phase out inconsistencies and reduce the use of in vivo models.
Modelling Alzheimer's disease using stem cells
Dr Eric Hill, Aston University, presented his research on brain hypometabolism in Alzheimer's disease progression during Research & Innovation 2016.
Currently, the majority of studies on Alzheimer's disease have used transgenic animal models or imaging studies of the human brain. It is difficult to validate these findings using human tissue. Whilst animal models have been central to our understanding of human physiology, human stem cell-based models may help us to further our understanding of human physiology and tackle devastating diseases such as Alzheimer's disease.
Dr Hill covers the following in this presentation:
- Brain hypometabolism is a major feature of Alzheimer's disease, appearing decades before cognitive decline and pathological lesions.
- Human stem cell-derived neuron and astrocyte cultures treated with oligomers of amyloid beta display a clear hypometabolism, particularly with regards to utilisation of substrates such as glucose, pyruvate, lactate and glutamate.
- As many neuronal functions, such as memory formation and protection from oxidative stress require energy formed from oxidative phosphorylation, these cells are at high risk of hypometabolism.
- Further research using models derived from iPSCs may elucidate the mechanisms associated with amyloid beta-induced hypometabolism and therefore expedite the discovery of novel biomarkers and mechanisms associated with disease progression.
Rising to the challenges of human iPSC-derived cells for tox & drug screening
We presented 'in the field' data on our portfolio of human iPSC and primary cells demonstrating their proven ease of use, reliability and consistency as meaningful drug discovery tools. Here, we overcome the challenges of cell line variability and address the needs for cell scale-up in assay campaigns.