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High-Throughput Screens

The lack of therapeutics for devastating CNS diseases and the high failure rate of promising new drugs in clinical trials argue for the need to improve drug discovery strategies. Improving clinical relevance along the drug discovery pipeline can increase the likelihood of clinical success. Using BrainXell’s patient iPSC-derived neurons in high-throughput screens for new therapeutics improves the clinical relevance at the initial stages of drug discovery. For example, spinal muscular atrophy (SMA) patient motor neurons were used in phenotypic screening for small molecules that can upregulate expression of endogenous SMN2 protein, a promising strategy for treating this fatal disease. More than a billion spinal motor neurons were produced to screen more than 500,000 analytes in 1536-well format. Such a large screen has never been performed using patient-derived neurons, and several technological innovations were developed to enable this high-throughput screen: 1) genetic engineering of patient derived iPSCs to generate a HTS compatible readout of SMN2 expression, 2) large and consistent production of neurons (10^9 motor neurons per batch), and 3) consistent plating of rapidly maturing neurons across hundreds of 1536-well plates. These innovations can be applied to other cortical or motor neuron disease drug discovery in an effort to improve clinical translatability (and success) at the earliest stage of drug discovery.

Electrophysiology of Neurons

Functioning neurons have precisely controlled membrane potentials and electrical currents flowing through ion channels. These electrical properties are integral to their role in communicating signals throughout the nervous system. Are you interested in studying pathphysiology, pharmacology, or the molecular and cellular processes that govern signaling? Electrophysiology techniques are used to experimentally probe the electrical properties of neurons. A variety of techniques are employed to directly or indirectly measure changes in electrical currents that flow across cell membranes or changes in the electrical potentials near the cell membranes.

Whole-cell recordings and patch recordings are obtained using specialized recording electrodes and amplifiers that make it possible to measure voltage changes while clamping the current, or measure currents while holding the voltage constant. These techniques allow for the study of ion channels, voltage sensitive proteins, and functional responses to neuro-active compounds. An example is shown of a whole cell recoding in current-clamp mode on BrainXell’s Spinal Motor Neurons (BX-0100) that exhibit spontaneous action potentials.

Whole-cell recordings and patch recordings are tedious techniques that require significant time for data acquisition. However, automated platforms are commercially available that offer improved throughput. As demonstrated here using a Qube 384 (Sophion), the electrophysiological properties of hiPSC-derived motor neurons produced by BrainXell can be characterized and Current-Voltage (IV) relationships for both sodium currents and potassium currents can be measured for dozens of cells.

The Qube 384 was also used to compare the phenotypes of SMA disease motor neurons to wild-type motor neurons. The NaV IV relationship of the SMA motor neurons displayed greater peak NaV current as compared to the control.

Extracellular recordings, which examine changes in electrical field potentials created by neurons that are adjacent to recording electrodes, can also be used to observe neuronal activity. This technique often utilizes Multi-Electrode Arrays (MEA) that record extracellular action potentials (spikes) from numerous neurons at the same time, allowing networks and synchronized signaling to be studied. Such recordings are non-invasive, examine the entire cell culture, and allow for repeated measurements to be made over time in the same plate.

MEA has also been used for functional phenotypic in vitro screening of patient iPSC-derived neurons.

Calcium Assays of Neurons

Functional activity of neurons is greatly influenced by prominent calcium signals that regulate neurotransmitter release and membrane excitability. Calcium signaling can be examined in a high-throughput manner with the use of fluorescent calcium-sensitive dyes. The relative fluorescence emitted by these dyes increases when bound to calcium and, once loaded into cells, provides a proxy for changes in cytoplasmic calcium concentrations. Image-based detection systems are available that support a wide range of formats including 96, 384, and 1536-well plates.

Changes in calcium concentration are closely tied to neuronal activity as action potentials are associated with large pre-synaptic calcium influx and a notable rise in postsynaptic calcium at excitatory synapses. This can be observed experimentally by chemical depolarization or electrical stimulation of neurons. Here, electrically evoked calcium responses are observed in the presence of various compounds.

Spontaneous calcium signals can also be observed in vitro when neurons are cultured under suitable conditions to form mature networks that exhibit spontaneous oscillations. Such oscillations are reflective of a population of neurons having synchronous network activity. This system can be used to screen pharmacological agents that alter neuronal activity, as demonstrated below using Brainxell’s Mixed Cortical Neurons (BX-0500).

Viability/Cytotoxicity Assays

Whether looking at toxicity of a compound, studying a disease phenotype, or exploring cellular mechanisms, assessing the viability of your cell culture can be a critical tool. A variety of cell viability assay kits are commercially available that are based on colorimetric or luminescent reagents, making readouts straightforward. There are also cytotoxicity assays that can assess cell membrane damage, such as an LDH assay, or measure apoptosis, such as a TUNEL assay. In this example, spinal motor neurons (BX-0100) cultured in 96-well plates were treated with 2 different compounds for 1 day and viability measured using a CellTiter-Glo® Luminescent Cell Viability Assay (Promega). This assay provides a luminescent signal that is proportional to the amount of ATP present in the culture. The amount of ATP, an indicator of metabolically active cells, is directly proportional to the number of living cells present in culture.

High-Content Analysis of Neurons

BrainXell’s neuronal products are widely used in high-content analysis (HCA), high-throughput screening, and large-scale automated live-cell imaging. HCA uses multi-parameter image processing and visualization tools to extract quantitative data from cell populations such as spatial distribution of proteins or cell structures.

HCA has been employed for screening of environmental toxins using iPSC-derived motor neurons that were gene-edited to ubiquitously express eGFP (Catalog number BX-0101). Following optimization of culture conditions in a 384-well format and imaging parameters for neuron number and neurite length, high-content imaging yielded a sensitive and robust system with a Z-prime value greater than 0.5.

The detection of neurons and neurites are demonstrated here. Original images (A) were processed to detect cell bodies (B) and neurites (C) for the control (DMSO) and 20 µM rotenone conditions. Dose-response curves for both neuron number (E) and total neurite length per well (F) were measured.

3-D Neural Spheroids

In order to develop the most relevant model systems for studying cell physiology, disease modeling, and drug discovery, many scientists are exploring three dimensional (3D) cultures. Such cultures provide a microenvironment, cell-to-cell interactions, and biological processes that may better represent in vivo conditions.

Technology

Overview
Our mission is to provide high quality neurons from human induced pluripotent stem cells (iPSCs) for CNS research and drug discovery. We feel strongly that using human iPSC-derived neurons increases the clinical relevance of neuroscience research programs. With iPSC-derived neurons scientists can study and model CNS diseases of clinical importance in the relevant genetic and cellular context. Our approach of directed differentiation yields high quality neurons in batch sizes of more than a billion neurons; enough to supply even the largest project demands. For example, we have used SMA patient-derived motor neurons in high-throughput phenotypic screens of more than 100,000 compounds, which is a scale beyond the scope of primary neurons.

Solid Foundation of our Technology
We direct human stem cells, including iPSCs, to subtype-specific neurons using proven technology developed by Prof. Su-Chun Zhang at the University of Wisconsin-Madison. [1-4]
Our directed differentiation protocols drive stem cells through cell fate determination stages observed during embryonic development. [4] This means our iPSC-derived neurons are more similar to primary neurons than others’ rapidly differentiated cells.

BrainXell’s Expertise
Our company focuses only on producing neurons and glia, which allows us to dedicate all of our energy manufacturing the purest cultures of physiologically-relevant mature neurons. With less than a dozen products, we can provide the best quality and support for each cell type. Our job is not done when a product is developed; we continue to meticulously optimize each differentiation step to yield a culture that performs reliably and expectedly.
Our production technology allows for massive expansion of cells during directed differentiation. Each batch of neurons yields at least a billion neurons–enough for a thousand 96-well plates! With large batch sizes, customers can expect reproducible results across multiple experiments or an entire high-throughput screening campaign.

Disease Modeling with iPSC-derived Neurons
Traditionally, biomedical research depended on the use of model systems to explore basic biology, probe disease mechanisms, and conduct drug discovery and development. However, results from such systems have low translatability, which limits the number of questions that can be addressed with such systems. [5] Therefore, a scientist should select a model system with the greatest physiological relevance that is technically and economically feasible for a given project. As technologies improve, more options become available to scientists to improve study design and increase the translatability of each experiment.
For early stage neuroscience and CNS drug discovery efforts, human neurons derived from iPSCs can add value to research programs. Human iPSC-derived neurons can replace non-human primary cells and immortalized human cell lines, which have limited predictive power to drive research in the right direction. In fact, scientists in our group have used patient iPSC-derived neurons to study spinal muscular atrophy (SMA) [6] as well as amyotrophic lateral sclerosis (ALS). [7] From these investigations, disease phenotypes were identified that helped inform ongoing drug discovery campaigns.

Videos

Brief introduction to BrainXell’s technology

Webinar introducing iPSC-Derived Human Neurons and their applications

References
Li, X.J., et al., Directed differentiation of ventral spinal progenitors and motor neurons from human embryonic stem cells by small molecules. Stem Cells, 2008. 26(4): p. 886-93. PMID: 18238853
Hu, B.Y., Z.W. Du, and S.C. Zhang, Differentiation of human oligodendrocytes from pluripotent stem cells. Nat Protoc, 2009. 4(11): p. 1614-22. PMID: 19834476
Du, Z.W., et al., Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun, 2015. 6: p. 6626. PMID: 25806427
Liu, H. and S.C. Zhang, Specification of neuronal and glial subtypes from human pluripotent stem cells. Cell Mol Life Sci, 2011. 68(24): p. 3995-4008. PMID: 21786144
Scannell, J.W. and J. Bosley, When Quality Beats Quantity: Decision Theory, Drug Discovery, and the Reproducibility Crisis. PLoS One, 2016. 11(2): p. e0147215. PMID: 26863229
Liu, H., et al., Spinal muscular atrophy patient-derived motor neurons exhibit hyperexcitability. Sci Rep, 2015. 5: p. 12189. PMID: 26190808
Chen, H., et al., Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons. Cell Stem Cell, 2014. 14(6): p. 796-809. PMID: 24704493