Name: rapid neuronal differentiation of induced pluripotent stem cells for measuring network activity on micro-electrode arrays
Uploaded: 2017-01-08T13:00:00.000+00:00
Duration: 9 min 20 s
Description: Watch this Scientific Journal Video about Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays at JoVE.com

The authors thank Jessica Classen for performing the whole-cell patch-clamp experiments. The hiPSCs used in our experiments were kindly provided by Huiqing Zhou and Willem van den Akker from the Radboud University Nijmegen. The transfer vectors used in this protocol were kindly provided by Oliver Br&#252;stle, Philipp Koch and Julia Ladewig from the University of Bonn Medical Centre.

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The authors have nothing to disclose.

Here we have implemented an efficient hiPSC-differentiation protocol published by Zhang et al. (2013)12 for measuring the network activity of hiPSC-derived neuronal networks on MEAs. We adapted the original protocol by creating an rtTA/Ngn2-positive hiPSC line before inducing neuronal differentiation. This additional step allows us to control the neuronal cell density on the MEA. Control over the neuronal density was an important pre-requisite for adapting the protocol to MEAs and for ensuring consistency. To measure the activity of neuronal networks using MEAs, the neurons need to form dense networks directly on top of the MEA electrodes17,18. This necessarily requires tight control over the plating density of the neurons. The rtTA/Ngn2-positive hiPSC line allows for control of neuron density because this tactic does not rely on acute lentiviral transductions of hiPSCs prior to differentiation; the rtTA/Ngn2-positive hiPSC line therefore nearly eliminates any variation in the final yield due to, for example, lentiviral toxicity and variable infection efficiency.
Another critical step of the experimental procedure is the number of the rat astrocytes that are cocultured with the differentiating hiPSCs. Astrocytes actively contribute to the refinement of developing neural circuits by controlling synapse formation, maintenance, and elimination, all of which are important processes for neuronal functioning. The protocol presented in this paper is highly astrocyte-dependent: to fully mature and form functional synapses, the neurons require support from the astrocytes. We experienced that the number of astrocytes should be roughly equal to the number of hiPSC-derived neurons to support the maturation of the neurons and the formation of neuronal networks exhibiting spontaneous activity. Since our astrocyte protocol yields primary cell cultures with a limited life span, the isolation of rat astrocytes has to be performed regularly.
Our adaptation of the protocol published by Zhang et al. (2013)12 for use with MEA technology will likely significantly improve our ability to study the network activity of hiPSC-derived networks. Previously, protocols used for studying hiPSC-derived neuronal networks with MEAs relied on time-consuming differentiation procedures13-16. The protocol from Zhang et al. (2013) provides a rapid alternative, and our modification removes a source of variability, which makes it now more feasible to use hiPSC-derived neurons in combination with MEA technology, especially in high-throughput or pharmacological studies. In addition, because the method published by Zhang et al. (2013)12 yields a homogeneous population of upper-layer cortical neurons, our adapted protocol makes possible focused studies into the network activity of this particular neuronal subset.
Nonetheless, this approach has also several limitations. First, the homogeneity of the cultures can also be considered a disadvantage, because the cultures are less likely to resemble in vivo networks, where different classes of neurons (i.e.&#160;inhibitory and excitatory neurons) constitute a heterogeneous network. To further enhance the use of the hiPSC-derived neurons with MEA technology, it will be important to develop rapid (transgene-based) differentiation protocols for other neuronal cell populations. If protocols become available, the in vitro networks would mimic in vivo networks more closely. Second, at present rat astrocytes must be added to the hiPSC-derived neurons for growth support, and therefore the resulting neuronal network is not a human neuronal network sensu stricto. Reliable protocols for differentiating hiPSCs into astrocytes may in the future solve this problem26. Third, two-dimensional neuronal networks, as described here, are a limited model for studying complex three-dimensional in vivo neuronal networks. Fortunately, protocols describing three-dimensional cultures of rat primary neurons in combination with MEA technology are already available27,28. Prospectively, the combination of rapid differentiation protocols for obtaining hiPSC-derived neurons and astrocytes with three-dimensional culture techniques and MEA technology should provide novel insight into the biological mechanisms underlying neurological disorders.

The development of human induced Pluripotent Stem Cells (hiPSCs) differentiation protocols to generate human neurons in vitro has provided a powerful new tool for studying neurological disorders. Until recently, the study of these disorders was severely hampered by the lack of model systems using human neurons. Although rodents can be used to study neurological disorders, the results of such studies cannot be translated easily to humans1. Given these limitations, hiPSC-derived neurons are a promising alternative model that can be used to elucidate molecular mechanisms underlying neurological disorders and for in vitro drug screening.
In the past decade, several protocols to convert hiPSCs into neurons have been developed2-8. However, these protocols are still limited in many ways. First, the protocols are often time-consuming: generating neurons with adequate maturation (i.e.&#160;synapse formation) and functional activity requires months of culturing procedures, which renders large-scale studies difficult9. In addition, hiPSC-to-neuron conversion efficiency is low. Protocols often yield a heterogeneous population of neurons, and thus do not allow studies of specific subsets of neuronal cells. Moreover, the protocols are not reproducible, yielding different results for different iPSC lines10,11. Lastly, the maturation stage and functional properties of the resulting neurons are also variable 10.
To address these problems, Zhang et al. (2013)12 developed a protocol that rapidly and reproducibly generates human neurons from hiPSCs by overexpressing the transcription factor neurogenin-2. As reported by the authors, differentiation occurs relatively quickly (only 2 - 3 weeks after inducing expression of neurogenin-2), the protocol is reproducible (neuronal properties are independent of the starting hiPSC line), and the hiPSC-to-neuron conversion is highly efficient (nearly 100%). The population of neurons generated with their protocol is homogeneous (resembling upper-layer cortical neurons), allowing the investigation of cell-type specific contributions to neuronal disorders. Furthermore, their hiPSC-derived neurons exhibited mature properties (e.g., the capability to form synapses and robust functional activity) after only 20 d.
Characterizing the electrophysiological properties of hiPSC-derived neurons at the network level is an important prerequisite before hiPSC technology can be exploited for the study of human diseases. For this reason, many research groups have recently begun to investigate stem-cell-derived neurons at the network level using micro-electrode array (MEA) devices (Multichannel Systems, Reutlingen, Germany)13-16. The electrodes of a MEA are embedded in a substrate on which neuronal cells can be cultured. MEAs can be used to explore the electrophysiological properties of neuronal networks and the in vitro development of their activity. Currently, MEAs are used only in combination with differentiation protocols that take several months to yield mature networks. Hence, combining MEAs with a rapid differentiation protocol should facilitate the use of this technology in large-scale studies of neurological disorders.
Here, we present a modification of the Zhang et al. (2013)12 protocol and adapt it for use on MEAs. In particular, rather than relying on an acute lentiviral transduction, we instead created hiPSC lines stably expressing rtTA/Ngn2 before inducing differentiation. We did this primarily to have reproducible control over the neuronal cell density, since the neuronal cell density is critical for neuronal network formation, and for good contact between the neurons and the electrodes of the MEA17,18. Although the Zhang et al. protocol is very efficient with respect to conversion of transduced hiPSCs, it is inherently variable with respect to the final yield of neurons from the number of hiPSCs plated initially (see Figure 2E in Zhang et al.)12. With a stable line, we eliminate many issues causing variability, such as lentiviral toxicity and infection efficiency. We then optimized the parameters that reliably produce hiPSC-derived neuronal networks on MEAs, obtaining network maturation (e.g., synchronous events involving a majority of the channels) by the third week. This rapid and reliable protocol should enable direct comparisons between neurons derived from different (i.e.&#160;patient-specific) hiPSC lines as well as provide robust consistency for pharmacological studies.

<table><tbody><tr><td>Lebovitz&#039;s L-15 medium</td><td>Gibco</td><td>11415-064</td><td></td></tr><tr><td>B-27 supplement</td><td>Gibco</td><td>0080085SA</td><td></td></tr><tr><td>Poly-D-Lysine (PDL)</td><td>Sigma-Aldrich</td><td>P6407</td><td></td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;-free HBSS&amp;nbsp;</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>0.05% Trypsin-EDTA&amp;nbsp;</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>2.5% Trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>High-glucose DMEM</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Sigma-Aldrich</td><td>F2442-500ML</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>psPAX2 lentiviral packaging vector</td><td>Addgene</td><td>Plasmid #12260</td><td></td></tr><tr><td>pMD2.G lentiviral packaging vector</td><td>Addgene</td><td>Plasmid #12259</td><td></td></tr><tr><td>Basement membrane matrix</td><td>Gibco</td><td>A1413201</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320-074</td><td></td></tr><tr><td>Cell detachment solution</td><td>Sigma-Aldrich</td><td>A6964</td><td></td></tr><tr><td>E8 medium</td><td>Gibco</td><td>A1517001</td><td></td></tr><tr><td>ROCK inhibitor</td><td>Gibco</td><td>A2644501&amp;nbsp;</td><td>Alternatively, ROCK inhibitors like thiazovivin can be used.</td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich</td><td>H9268-5G</td><td></td></tr><tr><td>G418</td><td>Sigma-Aldrich</td><td>G8168-10ML</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P9620-10ML</td><td></td></tr><tr><td>Vitronectin</td><td>Gibco</td><td>A14700</td><td></td></tr><tr><td>6-well MEAs</td><td>Multi Channel Systems</td><td>60-6wellMEA200/30iR-Ti-tcr</td><td></td></tr><tr><td>Glass coverslips</td><td>VWR</td><td>631-0899</td><td></td></tr><tr><td>Poly-L-Ornithine (PLO)</td><td>Sigma-Aldrich</td><td>P3655-10MG</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>Doxycyclin</td><td>Sigma-Aldrich</td><td>D9891-5G</td><td></td></tr><tr><td>N-2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Sigma-Aldrich</td><td>M7145</td><td></td></tr><tr><td>NT-3, human recombinant</td><td>Promokine</td><td>C66425</td><td></td></tr><tr><td>BDNF, human recombinant</td><td>Promokine</td><td>C66212</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>L-alanyl-L-glutamine</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Cytosine &amp;beta;-D-arabinofuranoside&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C1768-100MG</td><td></td></tr><tr><td>Straight fine-tipped forceps</td><td>Fine Science Tools</td><td>11251</td><td></td></tr><tr><td>Fine-tipped spring scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr></tbody></table>

rapid neuronal differentiation of induced pluripotent stem cells for measuring network activity on micro-electrode arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

Here we have successfully modified a protocol in which hiPSCs are differentiated directly into cortical neurons by over-expressing the transcription factor neurogenin-212 and we have adapted it for the use of MEAs. This approach is fast and efficient allowing us to obtain functional neurons and network activity already during the third week after the induction of differentiation.
During the course of the differentiation protocol, the cells morphologically started to resemble neurons: small processes were formed and neurons started connecting to each other (Figure 1A). We established a neurophysiological profile of the neurons derived from a healthy-control hiPSC line, by measuring their neuronal morphology and synaptic properties during development. hiPSC-derived neurons were stained for MAP2 and synapsin-1/2 at different days after the start of differentiation (Figure 2A). The derived neurons show mature neuronal morphology already 3 weeks after the induction of differentiation. The number of synapsin-1/2 puncta (a measure for the number of synapses) was quantified based on synapsin-1/2 immunocytochemistry stainings. The number of synapsin-1/2 puncta increased over time, suggesting that the level of neuronal connectivity is also increasing (Figure 2B). The number of synapsin-1/2 puncta 23 days after the induction of differentiation was similar in two independent IPS lines (Figure 2C). At 23 DIV most synapsin1/2 puncta were juxtaposed to PSD-95 puncta, which is indicative for functional synapses (Figure 2D).
Consistent with the results described by Zhang et al., we generated a population of excitatory upper layer cortical neurons, confirmed by pan-neuronal and subtype-specific cortical markers such as BRN2 and SATB2(layer II/III). We did not observe neurons that were positive for deep layer neurons CTIP2 (layer V) or Foxp2 (layer VI) (Figure 2E and&#160;F)
To characterize the electrophysiological activity of the hiPSC-derived neurons, we used whole-cell current and voltage clamp recordings, i.e.&#160;intrinsic properties and excitatory input onto these neurons were measured during development. The neurons were able to generate action potentials already one week after the of differentiation and the percentage of spiking cells was increasing over time (Figure 2G and&#160;H). Furthermore, the neurons received excitatory synaptic input already a week after the induction of differentiation: both frequency and amplitude of the excitatory synaptic input increased during development (Figure 2I - K).
To better understand how single-cell activity combines to form network-level functions, it is essential to study how neurons work in concert. In vitro neuronal networks cultured on MEAs constitute a valuable experimental model for studying the neuronal dynamics. We recorded 20 min of electrophysiological network activity of neurons derived from a healthy-control hiPSC line cultured on 6-well MEAs (Figure 2M). Few weeks after the induction of differentiation, the neurons derived from healthy-control hiPSCs formed functionally active neuronal networks, showing spontaneous events (0.62 &#177; 0.05 spike/s; Figure 2N). At this stage of development (i.e.&#160;16 d&#160;after the start of the differentiation) no synchronous events involving all the channels of the MEAs are detected (Figure 2O). The level of network activity increased during the development: during the fourth week after the induction of differentiation, the neuronal networks showed high level of spontaneous activity (2.5 &#177; 0.1 spike/s; Figure 2N) in all the wells of the device. The networks also exhibited synchronous network bursts (4.1 &#177; 0.1 burst/min, Figure 2O) with long duration (2,100 &#177; 500 ms).
<img alt="Figure 1" src="/files/ftp_upload/54900/54900fig1.jpg" /> 
Figure 1: hiPSC Differentiation into Neurons. A. Three time points of hiPSCs differentiation into neurons on coverslips. B. Plating of hiPSCs on MEAs. C. Astrocytes at 100% confluency in T75 flask (note that the cells form a tessellated monolayer). Scale bars: 150 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54900/54900fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54900/54900fig2.jpg" /> 
Figure 2. hiPSC-derived Neurons Characterization. A. hiPSC-derived neurons were stained for MAP2 (green) and synapsin-1/2 (red) at different days after the start of differentiation. Scale bar: 10 &#181;m. B. Quantification of synapsin puncta in two independent experiments. In each experiments at least 10 cells were analyzed. C. Quantification of synapsin puncta at DIV23 in neurons derived from two independent IPS lines. D. hiPSC-derived neurons were stained for PSD-95 (green) and synapsin-1/2 (red) 23 days after the start of differentiation. Synapsin puncta are juxtaposed to PSD-95 puncta. E. hiPSC-derived neurons were stained for MAP2 (green) and BRN2 (red) or SATB2 (red) 23 d after the start of differentiation. F. Percentage of MAP2 positive cells that were positive for indicated markers. G. Representative current clamp recordings showing that action potentials can be generated as early as 7 d after the start of differentiation. H. Percentage of cells at different days after the induction of differentiation that show one or more action potentials. I. Representative traces of excitatory postsynaptic currents (EPSCs) received by hiPSC-derived neurons at different days after differentiation. J. Frequency of excitatory postsynaptic currents during development. K. Amplitude of excitatory postsynaptic currents during development. L. Representative traces of EPSC recordings without (control) and with CNQX (CNQX). M. Neurons derived from one hiPSC line were cultured on a 6-well MEA and network activity is shown for hiPSC-derived neuronal networks 16 and 23 d after the induction of differentiation. The activity recorded from each well (sampling rate of 10 kHz) is indicated with a different color (5 min of the 20 min of recording are shown). N. Firing rate 16 and 23 d&#160;after the induction of differentiation. O. Bursting rate 16 and 23 d&#160;after the induction of differentiation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54900/54900fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Given the results, the quality of the resulting hiPSC-derived neurons can be assessed by making a neurophysiological profile of the cells. That is, three to four weeks after the start of the differentiation, the morphology, synapsin-1/2 expression and electrophysiology of the neurons can be assessed. At that time point, the hiPSC-derived neurons are expected to show a neuronal-like morphology, to be MAP2, synapsin/PSD-95 positive when performing immunocytochemistry, and to exhibit spontaneous electrophysiological activity (both at the single-cell and network level).

Watch this Scientific Journal Video about Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays at JoVE.com

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

26.8K Views.  Radboudumc.  We wijzigen en implementeren van een eerder gepubliceerd protocol beschrijft de snelle, reproduceerbare en efficiënte differentiatie van humane geïnduceerde pluripotente stamcellen (hiPSCs) in prikkelende corticale neuronen 12. Specifiek onze modificatie maakt regeling van neuronale celdichtheid en gebruik op micro-electrode arrays elektrofysiologische eigenschappen op netwerkniveau meten. 

Video: Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Neuroscience

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Bioengineering

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

Measuring the Electrophysiological Activity of Neuronal Networks Using Micro-Electrode Arrays

Source: Frega, M. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017).
The video demonstrates how to use a microelectrode array (MEA) to record the electrophysiological activity of neurons derived from human induced pluripotent stem cells (hiPSCs). Electrophysiological activity from a multiwell MEA containing a neuron-astrocyte co-culture is recorded to detect action potentials from multiple neurons in the network, confirming the successful differentiation of hiPSCs into neurons.

1. Differentiation of&#160;rtTA/Ngn2-positive hiPSCs to Neurons on 6-well MEAs and Glass Coverslips
NOTE:&#160;In this protocol, the details are provided ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

Source: Frega, M., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating excitatory cortical neurons from human-induced pluripotent stem cells (hiPSCs). Upon doxycycline induction, the hiPSCs overexpress neurogenin-2, a neural-specific transcription factor. By using specific media and co-culturing with astrocytes, this process facilitates the hiPSCs&#39; differentiation into neurons with synaptic connections.

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

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