Neural Progenitor Cells: Methods and Protocols

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  1. Neural Stem Cells
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  3. Optimized protocols to turn induced pluripotent stem cells into neural precursor cells
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  5. Neural progenitor cells derived from human induced pluripotent stem cells.

Culture rosette-derived NSCs in the presence of NI medium, refreshing the medium every other day until the cells reach confluence. When confluent, passage the NSCs as described in the following steps. Add 1. Rinse the Petri dish or T flask with an equal volume of NI medium 1. Further dilute the cell suspension in 3 or 4 mL of complete NI medium and count the cells using trypan blue and an automated cell counter. Centrifuge passaged NSCs from step 3. NOTE: The cells should be counted at step 3.

Aliquot the cells in suitable vials for cryopreservation about 0. NOTE: Upon differentiation, neuronal and glial derivatives can be characterized using different techniques, such as the ones described in the following sections. Run qPCR reactions in duplicate using appropriate master mix and primer sets see the Table of Materials. Alternatively, use another suitable method. Immunocytochemistry and high-content imaging HCI 6 , When ready for staining, permeabilize the cells in permeabilization buffer 1x DPBS containing 0.

Neural Stem Cells

Incubate the cells for 45 min at room temperature in blocking buffer containing fluorochrome-conjugated secondary antibodies see the Table of Materials , counterstaining the nuclei with DAPI dye. Quantify the mean fluorescence intensity and the relative percentages of cell types using a suitable high-content imaging platform, if available see the Table of Materials. Additionally, reverse phase protein array RPPA assays and analyses may be performed, as described in Reference 12 for a list of tested antibodies, see the Table of Materials. Electrophysiological measurements Differentiate the cells for 3 weeks in complete ND medium, refreshing the medium twice a week.

At the end of differentiation, seal the MEA chips with a semi-permeable membrane under a laminar flow hood to keep the cultures sterile for repeated measurements. Replace one of the electrodes with one ground reference, allowing for recordings from the remaining electrodes. Detect peaks from the MEA raw data using threshold limit of Undifferentiated hiPSCs should be round, with large nucleoli and without abundant cytoplasm.

The majority of the colonies should be characterized by a flat and tightly-packed morphology, indicative of an undifferentiated phenotype Figure 2A and Figure 2B. These results should be reproducible over passages. Figure 2.

Significance of optimizing methods for reliable and consistent generation of neuronal subtypes

C representative images of alkaline phosphatase-stained colonies 4X magnification. HiPSCs are pluripotent, meaning that they express three germ layer-related genes under appropriate conditions. To assess hiPSC pluripotency, it is possible to apply a common approach based on spontaneous EB formation, which induces the formation of the three germ layers Figure 3.

Assessment of Pluripotency by EB formation. A Representative phase-contrast image of EBs at day 1. IMRiPSCs can be differentiated into mixed cultures of post-mitotic neurons and glial cells following the steps summarized in Figure 1 and in the protocol sections.

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Upon rosette dissociation and replating onto laminin- or standard matrix-coated dishes or plates in the presence of complete ND medium, cells start undergoing differentiation into mixed cultures of neurons and glia, progressively forming clusters of neuronal cell bodies connected by bundles of neurites Figure 4C and Figure 4D. Similar results should be obtained when analyzing neuronal populations obtained by expanding rosette-derived NSCs and differentiating them into neurons and glia.

It is important to consider that the percentage of each cell type i. The induction of differentiation can also be assessed by the analysis of pluripotency-related markers e. This can also be confirmed through the analysis of gene expression by qPCR, which should indicate a decrease of Oct4 and Nanog and the upregulation of neuronal genes, such as neural cell adhesion molecule 1 NCAM1 and microtubule-associated protein 2 MAP2 ; the presynaptic gene, synaptophysin SYP ; and the post-synaptic gene, microtubule-associated protein tau MAPT , as shown in Figure 4I.

The analysis of spontaneous electrical activity, by means of MEA, is a valuable readout to assess the functionality of the neuronal network in differentiated hiPSCs. However, bursts are not observed. Figure 4. The graphs in I and J were modified from Reference 6. K Representative raster plot of IMRNSC-derived neurons the recording was carried out for a minimum of s; the vertical bars represent single spikes.

In the new toxicity testing paradigm, it is essential to define the molecular and cellular events occurring within a cell following exposure to a given toxicant. Commercially available arrays for the analyses of protein kinase gene expression can be used to compare undifferentiated hiPSCs versus differentiated cells.

Optimized protocols to turn induced pluripotent stem cells into neural precursor cells

Figure 5. Gene expression data were normalized to the reference genes 18S and GAPDH provided in the array and calibrated to undifferentiated cells. C The bar graph shows the absolute protein quantifications by means of RPPA analysis, comparing differentiated red bars and undifferentiated cells green bars. Rotenone, an inhibitor of complex I of the mitochondrial respiratory chain, is known to cause oxidative stress by triggering the activation of the Nrf2 pathway. Under quiescent conditions, Nrf2 is anchored in the cytoplasm by Keap1 Kelch-like ECH-associated protein 1 , an Nrf2 repressor, which facilitates Nrf2 ubiquitination and proteolysis Upon the induction of oxidative stress, Nrf2 translocates into the nucleus and activates the expression of Nrf2-ARE target genes IMRhiPSC-derived neurons and glial cells can be used to assess the effects of rotenone on Nrf2 activation by exposing the cells to different concentrations of rotenone e.

These concentrations were established according to previous studies 17 , Rotenone induced Nrf2 nuclear translocation, especially after exposing the cells to nM rotenone Figure 6B and Figure 6E.

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Figure 6. B Nrf2 protein nuclear translocation i. C Quantification of cytoplasmic Keap1 protein levels upon rotenone treatment, assessed by HCI analysis. E Representative images of Nrf2 protein localization green. Analogously, previous in vivo and in vitro studies have described a rotenone-dependent and selective dopaminergic neuronal cell death 21 , 22 , Figure 7.

Statistical significance was assessed by one-way ANOVA with Dunnett's Multiple Comparison Test as a post-test comparing all columns versus the control column 24 or by two-tailed unpaired or paired t-test according to the type of analysis. An asterisk over a bar indicates a significant difference with the control group.

Table 1.

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Note on dissociated rosette plating density: if rosette fragments do not look completely dissociated, in order to reach a cell plating density of about This work describes a robust and relatively rapid protocol for the differentiation of IMRhiPSCs into post-mitotic neurons and glial cells.

Previously published neuronal differentiation protocols based on hESCs and hiPSCs usually yield high percentages of neural precursors 25 , 26 and a significant number of neuronal target cells 27 , 28 , 29 , 30 , 31 , 32 , 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. The generation of EBs and neuroectodermal derivatives rosettes can be manually challenging and prone to variability. 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 NSCs 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's group 36 , 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 stress 38 , and oxidative stress is an important and common key event in various AOPs relevant to developmental and adult neurotoxicity 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.

Neural progenitor cells derived from human induced pluripotent stem cells.

It has been found that rotenone induces a dose-dependent increase of GFAP protein levels, indicative of astrocyte activation 40 , 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.

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 infections The authors would like to thank Dr.

Giovanna Lazzari and Dr. Tiziana Santini Italian Institute of Technology, Rome , for providing advice on immunofluorescence staining evaluation; Dr. Benedetta Accordi, Dr. Elena Rampazzo, and Dr. National Center for Biotechnology Information , U. J Vis Exp. Published online Jun 9. Author information Copyright and License information Disclaimer.