Name: protocol for the differentiation of human induced pluripotent stem cells into mixed cultures of neurons and glia for neurotoxicity testing
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The authors would like to thank Dr. Marc Peschanski (I-Stem, &#201;vry, France), for providing the IMR90-hiPSCs; Dr. Giovanna Lazzari and Dr. Silvia Colleoni (Avantea srl, Cremona, Italy); Dr. Simone Haupt (University of Bonn, Germany); Dr. Tiziana Santini (Italian Institute of Technology, Rome), for providing advice on immunofluorescence staining evaluation; Dr. Benedetta Accordi, Dr. Elena Rampazzo, and Dr. Luca Persano (University of Padua, Italy), for their contributions to the RPPA analysis and antibody validation. Funding: this work was supported by the EU-funded project "SCR&amp;Tox" (Grant Agreement N&#176;. 266753).

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EURL ECVAM Available from: <a target="_blank" href="https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/standard-operating-procedure-for-differentiation-of-human-induced-pluripotent-stem-cells-into-post-mitotic-neurons-and-glial-cells-%28mixed-culture%29-protocol-no.-165/key/p_1570">https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/standard-operating-procedure-for-differentiation-of-human-induced-pluripotent-stem-cells-into-post-mitotic-neurons-and-glial-cells-%28mixed-culture%29-protocol-no.-165/key/p_1570</a> (2016)</li><li>Standard operating procedure for expansion of rosette-derived neural stem cells. 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The authors have nothing to disclose.

This work describes a robust and relatively rapid protocol for the differentiation of IMR90-hiPSCs into post-mitotic neurons and glial cells. Previously published neuronal differentiation protocols based on hESCs and hiPSCs usually yield high percentages of neural precursors25,26 and a significant number of neuronal target cells27,28,29,30,31,32,33. Analogously, the differentiation protocol described here is suitable to generate heterogeneous cultures of GABAergic, glutamatergic, and dopaminergic neuronal cells, together with glia and a discrete proportion of nestin+ cells. The presence of glutamatergic (~35-42%) and GABAergic (~15-20%) neural cells suggests that this culture possesses forebrain, cortical-like features, and the presence of a discrete number of dopaminergic neurons (~13-20%) may also indicate midbrain specificity. Additionally, the permanence of a modest proportion of nestin+ cells may prove suitable for the study of neurogenesis and the possible effects of chemicals on NSCs, which are primarily confined to both the hippocampus and the subventricular zone (SVZ) of the forebrain34. Further immunocytochemical and gene expression analyses would help to better define the regional specificity of the differentiated cell derivatives.
The two most critical steps in the differentiation protocol described in this document are: (i) the cutting of hiPSC colonies into homogenous fragments (which is critical for the generation of EBs with homogenous sizes) and (ii) the cutting of neuroectodermal structures (rosettes) for NSC differentiation, which requires significant manual skill and precision to avoid collecting mesodermal and endodermal cells that may reduce the proportions of neurons and glial cells obtained upon differentiation.
It is crucial to characterize the phenotypes of the cells during expansion (as undifferentiated colonies or NSCs) and during all differentiation steps. In particular, the gene and protein expression profiles of the neuronal/glial cell derivatives should show upregulation and activation of neuron-related signaling pathways, whereas the expression of pluripotency markers should be decreased.
The generation of EBs and neuroectodermal derivatives (rosettes) can be manually challenging and prone to variability. For this reason, we developed a protocol for the expansion of rosette-derived NSCs and their further differentiation into neuronal/glial cells.
Possible limitations of this differentiation protocol are mainly (i) the relatively low percentage of differentiated glial derivatives and (ii) the lack of mature neuronal network functions (as shown by the lack of bursts). Moreover, specific subpopulations of astrocytes can function as primary progenitors or NSCs35. While nestin/GFAP double-positive cells were not observed in this differentiated cell culture (data not shown), it is hypothesized that the GFAP+ cells in these mixed cultures are astrocytic progenitors and astrocytes. It is plausible that by extending the time of differentiation, the number of astrocytes may increase, and their morphology may become more mature, as already indicated by previous works from Zhang&#39;s group36,37.
In the new toxicity testing paradigm, knowledge on chemical-induced perturbations of biological pathways is of the utmost importance when assessing chemical adversity. Therefore, in vitro test systems should be able to relate adverse effects to disturbances of signaling pathways, according to the concept of the adverse outcome pathway (AOP). As a proof-of-concept, rotenone can be used to assess the activation of the Nrf2 pathway, which is involved in the cellular defense against oxidative or electrophilic stress38, and oxidative stress is an important and common key event in various AOPs relevant to developmental and adult neurotoxicity39.
Rotenone should elicit the activation of the Nrf2 pathway, which can be demonstrated by Nrf2 protein nuclear translocation and increased expression of Nrf2-target enzymes, including NQO1 and SRXN1. It has been found that rotenone induces a dose-dependent increase of GFAP protein levels, indicative of astrocyte activation40,41. Rotenone also decreases the number of dopaminergic (TH+) cells, which is in agreement with previous in vitro and in vivo studies showing rotenone-dependent dopaminergic cell death, since this type of neuron is particularly sensitive to oxidative stress21,22,23.
In conclusion, this hiPSC-derived neuronal and glial cell culture model is a valuable tool to assess the neurotoxic effects of chemicals that elicit oxidative stress resulting in Nrf2 pathway activation. As this differentiation protocol allows for the generation of mixed cultures of neuronal cells (GABAergic, dopaminergic, and glutamatergic neurons) and astrocytes, it may prove suitable for studying the crosstalk between neurons and glia in physiological and pathological conditions, such as in neurodegenerative diseases (e.g., Parkinson&#39;s disease). Moreover, the presence of a significant proportion of NSCs may help to assess the possible effects of chemicals on neural progenitors, which are known to be the main target of chemically-induced mutations or viral infections42.

The US National Research Council report1 envisioned a new toxicity testing paradigm in which regulatory toxicity testing would be shifted from an approach relying on phenotypic changes observed in animals to an approach focusing on mechanistic in vitro assays using human cells. Pluripotent stem cell (PSC) derivatives may represent alternatives to cancer cell models, as the obtained cells may more closely resemble the physiological conditions of human tissues and provide more relevant tools to study chemical-induced adverse effects. The two major types of PSC cultures that are most promising for toxicity testing are human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), which are currently widely used in the areas of basic research and regenerative medicine2,3. This expertise can now be harnessed for the development of a new class of toxicological in vitro tests aimed at identifying the perturbed physiological pathways involved with the development of adverse effects in vivo. However, test methods for regulatory safety assessments based on hESCs would be unlikely to be accepted by all EU Member States and by countries worldwide due to possible ethical concerns and diverse national legislative policies regulating the use of embryo-derived cells.
hiPSCs share characteristics similar to hESCs4,5 and hold great potential for in vitro methods, both for identifying therapeutic targets, as well as for safety assessments. In addition, hiPSC technology mitigates the constraints of a limited donor pool and the ethical concerns associated with embryo-derived cells. A major challenge for hiPSCs is the demonstration that these cells can reproducibly generate a significant range of toxicologically relevant cell derivatives, with characteristics and responses typical of human tissues. Predefined levels of the selected markers are generally used to characterize cell populations after the differentiation process and to provide insights into the stability of the differentiation process.
Previous works evaluated the suitability of hiPSCs to generate mixed cultures of neuronal and glial cells and to assess the effects of rotenone, an inhibitor of mitochondrial respiratory complex I, on the activation of the Nrf2 pathway, a key regulator of anti-oxidant defense mechanisms in many cell types6,7.
This work describes a protocol used for the differentiation of hiPSCs into mixed neuronal and glial cultures, providing details on the signaling pathways (gene and protein level) that are activated upon neuronal/glial differentiation. Additionally, the work shows representative results demonstrating how this hiPSC-derived neuronal and glial cell model can be used to assess Nrf2 signaling activation induced by acute (24-h) treatment with rotenone, allowing for the assessment of oxidative stress induction.
IMR90 fibroblasts were reprogrammed into hiPSCs at I-Stem (France) by the viral transduction of 2 transcription factors (Oct4 and Sox2) using pMIG vectors6. Analogous hiPSC models can also be applied. The protocols described below summarize all the stages of differentiation of hiPSCs into neural stem cells (NSCs) and further into mixed cultures of post-mitotic neurons and glial cells (steps 1 and 2, also see the EURL ECVAM DBALM website for a detailed description of the protocol)8.
An additional protocol for the isolation, expansion, cryopreservation, and further differentiation of NSCs into mixed neurons and glial cells is detailed in steps 3 and 4 (also refer to the EURL ECVAM DBALM website for a detailed description of this protocol)9. Step 5 describes the analyses that can be done to assess the phenotypic identity of the cells during the several stages of commitment and differentiation.

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.

Watch this Scientific Journal Video about Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing at JoVE.com

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

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 ...

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