Human-induced pluripotent stem cell (iPSC)-derived neural cells offer a potent tool that can be employed to model disease pathology and neuronal behavior. The growing use of these cells in drug discovery helps in speeding up existing drug screening processes and decreases the use of in vivo models used at the initial phases of testing.
Notably, the production of particular populations such as cortical neurons has let scientists examine the activity of neural networks from specific areas of the brain. Adult cells from human individuals that contain disease-associated gene mutations can be reprogrammed into iPSCs and can subsequently be differentiated into a range of cell types such as cerebral cortical neurons (hCCNs) and human neural stem cells (hNSCs). The researchers aimed to phenotypically analyze patient iPSC-derived neurons containing the presenilin-1 (PS-1) mutation (L286V), and compare them with cells from healthy controls.
Characterization of Human Neural Stem Cells and Cerebral Cortical Neurons
Human Neural Stem Cells
Axol hNSCs form neural rosettes and express markers typically observed in neural precursor cells as seen by immunocytochemistry.
Human Cerebral Cortical Neurons
Differentiation of hNSCs generates hCCNs that express neuronal markers observed using immunocytochemistry. These neurons increase in maturity over time in culture.
Integration-free iPSCs were generated using an episomal vector and subsequently differentiated into hNSCs using Axol’s proprietary method.
Properties of iPSC-Derived Neurons and Astrocytes
(A)Voltage responses elicited by current steps in a patch clamped neuron, (B) current responses in an astrocyte elicited by voltage steps, and (C) network properties of iPSC-derived neurons and astrocytes. Electrical activity in maturing networks recorded on MEAs. Changes in the rate of depolarization spikes increase over time. These changes can be quantified as measures of network maturity and can be used to generate entropy-based connectivity maps between areas of the networks. (D) Field of hNSC in culture showing recording pipette, (E) fluorescence image showing the Fluo-4 loaded neurons, and (F) traces of fluorescence over time (from neurons circled in E) showing responses to GABA and glutamate. The trace below displays current from single neuron recorded from pipette shown in D.
Researchers created several endpoint assays using hNSCs (Axol Bioscience) to find out the functionality of these cells and their response to toxins or disease-relevant biomarkers in epilepsy as well as in Alzheimer’s disease.
Cell Culture hNSCs were received from Axol Bioscience and were differentiated using neuronal maintenance media. Cells culture was maintained for up to 12 months.
Patch Clamp recordings were done with the help of pipettes (2–4 MΩ) with an internal solution of the following composition (in mM): HEPES 10, KMeSO4 120, EGTA 0.1, GTP 0.5, and Na2ATP 4. A Multiclamp700b amplifier was used to record currents.
MEA Analysis: Cells were cultured on multi-electrode array dishes (Scientifica) and variations in the rate of depolarization spikes over time were measured and used to create entropy-based connectivity maps between the regions of the networks. The use of multi-well MEA is viable (Axion) to improve the throughput of the analysis.
Purification and Labeling of Recombinant TAU: Recombinant TAU was found in E. coli BL21™ cells and was decontaminated using Ni-affinity chromatography. Purification tags were eliminated by digesting it with TEV protease and the purified protein labeled with the thiol-reactive dye Atto 488 Malemide (Sigma UK). For uptake experiments, 1-µM labeled TAU was incorporated into the medium of differentiated cells.
(A) Release of amyloid peptides from patient ax0112 (presenilin-1 L286V) compared to “normal” control (ax0016). (B) Schematic representation of prokaryotic expression constructs used to obtain purified recombinant TAU labeled with Atto 488 Malemide visualized by SDS-PAGE. (C, D) Endogenous TAU production at different stages of neuronal differentiation. (E) Fluorescent calcium imaging of epileptiform activity in 4AP treated iPSC-derived cortical cultures. Following treatment with 100-µM 4AP, cells were treated with increasing doses of the anti-epileptic drug VPA (2 mM and 5 mM).
High-Throughput MEA Analysis
Simultaneous analysis of 12- or 48-well MEAs with 3 recording stations (Maestro, AxionBiosystems) allows high-throughput analysis of human iPSC cultures and test compound effects.
Axol iPSC Neural Stem Cells can be Differentiated into Neurons on MEAs
Summary and Conclusions
The use of iPSC-derived neural cells has allowed the investigation of network maturation and neuronal development in physiologically relevant human models. Furthermore, such models allow the examination of mature cultures of neurons/astrocytes in healthy models as well as those carrying disease-associated mutations.
The use of these models received as neural stem cells, which usually differentiate into functional neuronal networks, has accelerated the production of the preliminary data mentioned in this article. Advancing this study ahead by exploiting higher throughput MEA applications will certainly offer the platform to quickly expedite the understanding of brain function and pathology.
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.