Neural cells derived from induced pluripotent stem cells (iPSCs) serve as a robust tool for modeling neuronal behavior as well as disease pathology.
These cells are used extensively in drug discovery, which could help speed up the existing drug screening processes and thus lower the use of in vivo models that are utilized at the earliest phases of testing. Most significantly, the production of cortical neurons, dopaminergic neurons, and other specific populations has enabled scientists to study the activity of neural networks from specific parts of the brain.
As such, several end-point assays were developed using human iPSC-derived neural stem cells (Axol Bioscience) to establish the functionality of these cells as well as their response to disease-relevant biomarkers or toxins in both epilepsy and Alzheimer’s disease. The cells were also manipulated using Lentivirus, and the long-term expression of more than 9 months was demonstrated.
These techniques provide a platform to gain a better understanding of the normal physiological functions and also the causes of the pathology of the central nervous system (CNS).
Figure 1. Cortical differentiation of hiPSC cells. (A) Schematic of neuronal differentiation. (B) Immunofluorescent image of 14-day old cortical neurons and astrocytes B-tubulin (green)/ Neurofilament (Blue)/GFAP (red).
Figure 2. Lentiviral transduction of differentiated cultures for long-term expression. A) GFP expression in neurons and astrocytes 100 days post-transduction with lentivirus encoding GFP under the control of the CMV promoter (MOI 5). B) Neuronal expression of the channelrhodopsin (ChR2-E123T-T159C-EYFP) under the control of the CamKii promoter in 50-day old differentiated cultures, 9 days post-transduction and inset in 180-day old differentiated cultures 80 days post-transduction (MOI 5).
Figure 3. 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 network measures of maturity and can be used to generate entropy-based connectivity maps between areas of the networks.
Figure 4. Properties of iPSC-derived neurons and astrocytes. (A) Immunofluorescent image of cortical neurons. (B) Immunofluorescent image of pure astrocytes (C) Voltage responses elicited by current steps in a patch clamped neuron. D) Current responses in an astrocyte elicited by voltage step.
Figure 5. (A) Field of hNPCs in culture showing recording pipette. (B) Fluorescence image showing the Fluo-4 loaded neurons. (C) Traces of fluorescence over time (from neurons circled in (B) showing responses to GABA and glutamate. The trace below displays current from single neuron recorded from pipette shown in A.
Figure 6. Modeling Alzheimer’s disease using iPSC-derived cultures. (A) Release of Amyloid peptides from patient AX0112 (Presenilin-1 L286) 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) Uptake and spread of labeled K-18 in 24 hours after seeding media of differentiated neuronal cultures. (E) Endogenous Tau production at different stages of neuronal differentiation.
Figure 7. 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).
Neuronal maintenance media were used for differentiating the hNPC cells acquired from Axol Biosciences (UK). Cells were preserved in culture for a period of 12 months. Patch clamp recordings were subsequently made using pipettes (2–4 MΩ) with an internal solution of composition (in mM)—Na2ATP 4, KMeSO4 120, EGTA 0.1, HEPES 10, and GTP 0.5. A Multiclamp700B amplifier was used for recording currents.
Cells were cultured on multi-electrode array dishes (Scientifica), and changes in the rate of depolarization increased over time. These changes were measured and used for producing entropy-based connectivity maps between the areas of the networks.
To transduce the differentiated cultures, purified virus in PBS (at specified MOIs) was added directly to the media. Following overnight incubation, the cells were washed in neural maintenance medium to remove the excess virus.
Purification and Labeling of Recombinant Tau
After the recombinant Tau was expressed in E. coli BL21™ cells, it was purified using Ni-affinity chromatography. Subsequently, purification tags were removed through digestion with TEV protease and the resultant purified protein was labeled with Atto 488 Malemide—a thiol-reactive dye from Sigma UK. Finally, 1 μM-labeled Tau was introduced to the medium of the differentiated cells for uptake experiments.
In human-relevant models, the application of iPSC-derived neuronal models has enabled the analysis of the development of neurons as well as the maturation of the neuronal/neuronal network. Models like those also make it possible to examine mature cultures of astrocytes/neurons in healthy models and also in those bearing disease-associated mutations.
The use of these models as neural precursor cells, which normally differentiate into functional networks, has accelerated the creation of the preliminary data demonstrated here. This will also enable rapid testing and development of innovative methods. For example, the use of emerging technologies such as next-generation sequencing, genome editing¸ and high-throughput image/activity analysis in tandem with such models, will clearly offer the platform to quickly expedite one’s understanding related to function and pathology of the brain.
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.
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