Name: Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs) (Video) | JoVE Journal
Uploaded: 2018-03-02T13:30:00.000+00:00
Duration: 10 min 47 s
Description: Learn rapid phenotypic profiling in human neural precursor cells for neurodevelopmental research. This reproducible protocol assesses growth factor effects and cellular abnormalities in neuropsychiatric disorders.

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5 minutes

This work was supported by the New Jersey Governor&#39;s Council for Medical Research and Treatment of Autism (CAUT13APS010; CAUT14APL031; CAUT15APL041), Nancy Lurie Marks Family Foundation, Mindworks Charitable Lead Trust, and the Jewish Community Foundation of Greater MetroWest NJ.

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The authors declare that they have no competing financial interests.

The protocols presented here illustrate quick and simple methods to study fundamental neurodevelopmental processes and test growth factors and drugs using hiPSC-derived neural precursor cells. hiPSC technology has revolutionized the study of the pathogenesis of neurodevelopmental diseases by providing us with unprecedented access to live human neuronal cells from affected individuals. Indeed, there have been numerous hiPSC studies of neurodevelopmental disorders including Rett Syndrome, Timothy Syndrome, Fragile-X syndro...

The use of simpler organisms and mouse models has elucidated the mechanisms of basic brain development as well as disease pathogenesis. Despite these advances, the etiology of many neuropsychiatric disorders remains elusive because not all findings in simpler organisms are directly relevant to complex aspects of human disease. Further, the greater complexity of the human brain often makes it difficult to model human development and disorders in animals. With the evolution and progress of human induced pluripotent stem cells (hiPSCs) technology, somatic cells can be reprogrammed into stem cells and then differentiated into neuronal cells to study human disease. Advances in hiPSCs and &#34;omic&#34; technologies (genomics, transcriptomics, proteomics, metabolomics) promise to revolutionize the understanding of human brain development. These technologies now make possible a &#34;precision medicine&#34; approach to the characterization of neuropsychiatric disease on a case-by-case basis.
The current staple in the hiPSC disease-modeling field is to differentiate cells into specific neuronal subtypes in a monolayer or to use a 3D culture system called an organoid to recapitulate aspects of brain development1,2,3. These systems have been incredibly valuable in studying and uncovering unique aspects of human development and disease4,5,6,7. However, both neuronal cultures and organoids often require anywhere from weeks to months in culture before they are ready to study. The time-consuming nature of these protocols and the amount of resources needed to maintain these culture systems often limit the number of experiments that can be performed and the number of variables (like growth factors or drugs) that can be tested. Moreover, many studies utilizing post-mitotic neurons and organoids have focused on processes such as dendrite outgrowth or synapse formation, which occur later in development. While these processes have been implicated in the pathology of developmental disorders such as autism and schizophrenia, earlier developmental events that occur before definitive neuronal differentiation are also important for disease pathogenesis8,9,10,11,12,13. Indeed, recent genomic studies show that the mid-fetal period, which is comprised of proliferation, process outgrowth, and migration, is particularly important in autism pathogenesis11,14. Thus, it is important to study neural stem and progenitor cell populations to better understand these earlier processes. Organoid systems, which are considered to better recapitulate human brain development because of their 3D nature and organized structure, do contain a progenitor pool that has been utilized to study some of these earlier events. However, the progenitor population in organoids is often sparse and more like radial glial cells than neural stem or progenitor cells5,15. Thus, it would be beneficial to have a high throughput method to study early stages of neurodevelopment in an actively proliferative cell population.
In the lab, we have created a protocol that uses hiPSC-derived neural precursor cells (NPCs), a mixed population of neural stem and progenitor cells that is highly proliferative, to study neurodevelopmental processes such as proliferation, cell migration, and initial process (neurite) extension. These assays were developed from techniques used in our lab for decades to successfully study neurodevelopment in rat and mouse cortical cultures16,17,18,19,20,21,22,23. Importantly, it was also shown that phenotypes and regulatory signals defined in the rat and mouse culture systems are highly predictive of mechanisms that are active in vivo, indicating the value of these techniques16,17,18,19,24. After initial differentiation of hiPSCs to NPCs, these methods allow us to study vital developmental processes in a matter of days. These methods have many advantages: (1) they require little sophisticated equipment and are easy to implement, (2) numerous experimental replicates can be conducted in a short period of time, allowing for rapid confirmation of the reproducibility of results, and (3) culture variables such as coating matrices, effects of growth factors, and activity of drugs can be tested quickly and cost-effectively. Furthermore, we take advantage of the well-established role of extracellular growth factors as critical regulators of diverse developmental processes. NPCs were exposed to select developmental signals that directly stimulate events like proliferation, neurite outgrowth, and cell migration, and have found they enhance the ability to identify defects that are not apparent in control conditions19,25,26,27,28. Likewise, the ease of assessing drugs provides a powerful avenue to adopt precision medicine techniques to test the efficacy of various therapeutic interventions. Thus, this protocol facilitates a high throughput, reproducible, and straightforward methodology to study early brain development, disease pathogenesis, and the potential beneficial effects of growth factors and drugs on neurodevelopmental phenotypes.

<table><tbody><tr><td>PSC Neural Induction Medium:&lt;br/&gt; Protocol Link: https://goo.gl/euub7a&amp;nbsp;</td><td>ThermoFischer Scientific</td><td>A1647801</td><td>This is a kit that consists of Neurobasal (NB) medium and a 50x Neural Induction Supplement (NIS). The NIS is used to make 1X Neural Induction Medium and 100% Expansion Medium</td></tr><tr><td>Advanced DMEM/F12 Medium</td><td>ThermoFischer Scientific</td><td>12634-010</td><td>Component of 100% Expansion Medium</td></tr><tr><td>Neurobasal Medium</td><td>ThermoFischer Scientific</td><td>21103049</td><td>Component of both NIM and 100% Expansion Medium&amp;nbsp;</td></tr><tr><td>hESC-qualified Matrigel</td><td>Corning</td><td>354277</td><td>hESC-qualified extracellular matrix-mimic gel (ECM-mimic gel)&amp;nbsp;</td></tr><tr><td>Y-27632 (2HCl), 1 mg</td><td>Stem Cell Technologies</td><td>72302</td><td>ROCK inhibitor</td></tr><tr><td>6 well plates</td><td>Corning</td><td>COR-3506</td><td>Polystyrene plates used for NPC maintenance and for Neurosphere Migration Assay&amp;nbsp;</td></tr><tr><td>24 well plates</td><td>ThermoFischer Scientific</td><td>2021-05</td><td>Polystyrene plates: Used for NPC DNA Synthesis Assay</td></tr><tr><td>35 mm dishes</td><td>ThermoFischer Scientific</td><td>2021-01</td><td>Polystyrene plates: Used for NPC S-Phase Entry and Neurite Assay</td></tr><tr><td>Natural Mouse Laminin</td><td>Invitrogen</td><td>23017-015</td><td>Substrate for coating plates: Used for NPC DNA Synthesis, S-Phase Entry, and Cell Number Assays</td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141</td><td>Substrate for coating plates: Used for Neurite Assay&amp;nbsp;</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P0899</td><td>Substrate for coating plates</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFischer Scientific</td><td>15140122</td><td>Antibiotic, component of NIM, 100% Expansion and 30% Expansion Media&amp;nbsp;</td></tr><tr><td>StemPro Accutase</td><td>Gibco</td><td>A11105-01</td><td>1X Cell Detachment Solution&amp;nbsp;</td></tr><tr><td>2.5% Trypsin (10X)</td><td>Gibco</td><td>15090-046</td><td>10X enzymatic solution</td></tr><tr><td>0.5 M EDTA</td><td>ThermoFischer Scientific</td><td>AM9261</td><td>used in trypsin solution for lifting cells for DNA synthesis assay</td></tr><tr><td>tritiated [&lt;sup&gt;3&lt;/sup&gt;H]-thymidine</td><td>PerkinElmer</td><td>NET027E001</td><td>Radioactive tritium, thymidine</td></tr><tr><td>Fisherbrand 7 mL HDPE Scintillation Vials</td><td>Fisherbrand</td><td>03-337-1</td><td>Vials for liquid scintillation counting</td></tr><tr><td>EcoLite(+)</td><td>MP Biomedicals</td><td>0188247501&amp;nbsp;</td><td>Liquid scintillation cocktail</td></tr><tr><td>LS 6500 multi-purpose liquid scintillation counter</td><td>Beckman Coulter</td><td>8043-30-1194</td><td>Liquid Scintillation Counter</td></tr><tr><td>Skatron Semi-automactic Cell Harvester Type 11019</td><td>Molecular Devices &amp;amp; Skatron Instruments, Inc.</td><td></td><td>Semi-automatic cell harvester</td></tr><tr><td>Click-iT EdU Alexa Fluor&amp;reg; 488 Imaging Kit</td><td>ThermoFisher Scientific</td><td>C10337</td><td>EdU and staining kit for S-Phase Entry Assay</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>ThermoFisher Scientific</td><td>15250061</td><td>Assessing viability of cells</td></tr><tr><td>Grade GF/C filter paper</td><td>GE Healthcare Life Sciences, Whatman</td><td>1822-849</td><td>Glass fiber filter paper</td></tr><tr><td>Human Basic FGF-2</td><td>Peprotech</td><td>100-18B</td><td>growth factor</td></tr><tr><td>Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38)</td><td>BACHEM</td><td>H-8430</td><td>neuropeptide</td></tr></tbody></table>

rapid detection of neurodevelopmental phenotypes in human neural precursor cells (npcs)

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

One goal of these studies is to define the proliferative activity of the NPCs, that is, an increase in cell numbers. This is achieved by assessing DNA synthesis of the total cell population, a high-throughput approach that measures the incorporation of radioactive tracer tritiated thymidine into cell extracts, and reflects all cells engaged in S-phase, whether they are synthesizing for 5 minutes or the entire two hours. Additionally, these assays allow the determination of the proportion ...

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

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Research

JoVE Journal

Developmental Biology

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described.  Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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.

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

This video demonstrates the neural precursor cell neurosphere migration assay—an in vitro assay to study the migratory response of neural precursor cells from the neurosphere—via microscopic analysis.

NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere

This video demonstrates a method to differentiate human neural progenitor cells (NPCs) into neurons. The NPCs are introduced onto an established mouse astrocyte-rat cortical neuron co-culture, promoting NPC differentiation into neurons. The differentiation is confirmed by visualizing the cells under a confocal microscope.

A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

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.

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

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Learn rapid phenotypic profiling in human neural precursor cells for neurodevelopmental research. This reproducible protocol assesses growth factor effects and cellular abnormalities in neuropsychiatric disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

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