ISSCR 2016

The Annual Meeting of the International Society of Stem Cell Research (ISSCR) took place from 22 - 25 June in San Francisco, CA. View our posters and presentation below.


Posters

Serum-Free Human iPSC-Derived Cardiomyocytes for in vitro Testing

Using defined factors Oct3/4, KLF4, Sox2 and c-Myc, adult cells from healthy and patient donors can be reprogrammed to generate induced pluripotent stem cells (iPSCs). Subsequently, these can be differentiated into a variety of cell types including cardiomyocytes. Human iPSC-derived cardiomyocytes (iPSC-CMs) can be cultured in vitro under serum-free conditions and as such, offer a platform to investigate the effect of growth factors, cytokines and drugs on the development and functionality of human cardiomyocytes in vitro. Following differentiation, spontaneously beating iPS-CM’s were evaluated in 2D and 3D culture. Expression profiling by immunohistochemistry confirms the expression of cardiomyocyte selective markers including α-actinin, myosin heavy chain, atrial and ventricular myosin light chains, troponin-T and -I, β-catenin, vimentin, L-type calcium channels, connexin-40 and -43, telethonin and ankyrin repeat domain-1 (ANKRD1). Immunohistochemistry findings were validated by Western Blot for α-actinin and cardiac troponin-T expression. Additional analyses conducted, include bi-nucleate cell counts, cell form factor measurements and a comparison of plating efficiencies across a variety of substrates. The electrical activity of the iPSC-CMs was confirmed using a multi-electrode array (MEA), and the calcium dye Fluo4. One application of these cells is drug toxicity testing. To show proof of principle that this can be undertaken in a contactless manner using only genetically encoded tools, which offers several advantages compared to low throughput contact based methods with chemical dyes, we developed a simultaneous optical control/calcium imaging approach to replace the need for electrode stimulation and dyes. We are able to control the beat frequency of iPSC- CMs across the physiological range (0.3Hz – 2Hz) and can observe the anticipated effects of compounds such as Dofetilide, a known hERG inhibitor. Here, we have identified a range of characteristics in these human iPSC-CMs that confirms their ability to function as a highly-pure population of single beating human cardiomyocytes in vitro and presented evidence of a technically simple and scalable platform for cardiotoxicity screening assays.

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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.

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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.

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Induction of Plasticity Phenomena in Human Induced Pluripotent Stem Cell-Derived Cortical Neurons

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.

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Functional Maturation and Drug Responses of Human Induced Pluripotent Stem Cell-Derived Cortical Neuronal Networks in Long-Term Culture

The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20–30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

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Presentations

Electrophysiological Maturation and Pharmacological Responses of Human Induced Pluripotent Stem Cell-Derived Cortical Neuronal Networks in Long-Term Culture

The functional network of human induced pluripotent stem cell-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20–30 weeks. Neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. We also attempted the induction of long- term potentiation (LTP) and long-term potentiation depression (LTD) phenomena using MEA systems. High-frequency stimulation 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. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening.

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