Name: Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark (Video) | JoVE Journal
Uploaded: 2018-01-26T13:00:00.000+00:00
Duration: 10 min 9 s
Description: Learn to isolate and culture neural progenitor cells from cerebellar tissue for epigenetic analysis using ChIP. This method enables study of H3K79me2 histone modifications during brain development.

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We thank Henriette Bertemes for helping to establish the CGN cultivation protocol within the lab. This method paper was supported by the DFG-funded CRC992 Medical Epigenetics by funding to TV. The authors acknowledge the support of the Freiburg Galaxy Team: Pavankumar Videm, Bj&#246;rn Gr&#252;ning and Prof. Rolf Backofen, Bioinformatics, University of Freiburg, Germany funded by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and German Federal Ministry of Education and Research (BMBF grant 031 A538A RBC (de.NBI)).

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</ol>

The authors declare that they have no competing financial interests

There are two major ways to perform chromatin immunoprecipitation to detect the genomic occupancy of histone modifications, transcription factors, histone code readers, writers, or erasers. One is the native ChIP method using nuclease digested, native chromatin for immunoprecipitation, and the other is the presented method using PFA-fixed, sheared chromatin, in which the nucleosomes and other DNA-attached proteins are covalently bound to the DNA39. Native ChIP with its high antibody detection rate...

The sensory, motor, and cognitive functions of the brain are highly complex and susceptible to physical and environmental changes. The brain consists of three general parts the hind-, mid-, and forebrain, which are deeply connected. Within the forebrain, the telencephalon can be divided into a dorsal telencephalon (DT) and a ventral telencephalon (VT). The DT of mice consists of six cortical layers which are formed between E11.5 and E18.5 in an &#34;inside-out&#34; manner1. The VT includes the ganglionic eminences in development, which later form the basal ganglia2,3. Several cell types can be classified in the mammalian central nervous system such as neurons, astrocytes, or oligodendrocytes4, which develop in a temporo-spatial manner5. First, the neural progenitor cells (NPCs) give rise to different kinds of neurons, interneurons in the VT, and projection neurons in the DT, and later on to glial cells (e.g., astrocytes6). During cortical development, the most superficial layer (layer I), which contains Cajal-Retzius cells, is formed first. Then, between E12.5 and E14.5, NPCs generate deeper neuronal layers (VI, V) while between 14.5 and 16.5, progenitors give rise to upper layer (IV-II) neurons7,8. Neuronal identity is specified by different morphogen-induced temporo-spatial transcriptional programs and additionally by epigenetic programs2.
The cerebellum, which is implicated in motor coordination, is located in the hindbrain and develops between E10 and roughly P20 in mice9. It contains the cerebellar cortex and the cerebellar nuclei10. The adult cerebellar cortex consists of three layers, the outermost molecular layer, the Purkinje cell layer, and the innermost granular layer containing granular neurons10. The cerebellar granule cells are the smallest neurons and represent about 80% of all neurons in the vertebrate brain11. They develop from precursors located in the external germinal zone and migrate through the Purkinje cell layer to their destination12. Like in the telencephalon, the development of the cerebellum is regulated by several important morphogens, which have specific time- and space-dependent functions and initiate defined transcriptional programs10.
The development of cortical and cerebellar layers is controlled by transcriptional expression of specific morphogens and, thus, by the chromatin state of the DNA. In a simplified view, chromatin states can be divided into euchromatin as transcriptionally active and heterochromatin as transcriptionally silent regions. The nucleosome as the basic unit of chromatin contains two copies of each core histone H2A, H2B, H3, and H4, surrounded by 147 base pairs of DNA13. Histones are highly post-translationally modified by methylation, acetylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, deamination, and proline isomerization14,15. Histone lysine methylation is considered to be the most stable histone modification that controls transcription, replication, recombination16, DNA-damage response17, and genomic imprinting18. Lysines can be mono-, di-, or tri-methylated19 and appear not only on the accessible histone tails but also within the globular domain of histones20. Specific methylations at H3K4 and H3K36 are mainly associated with euchromatin, specific methylations at H3K9, H3K27, or H4K20 are mainly found in heterochromatic regions, although all residues are located within the histone tail14,19,21. H3K79 methylation is located within the histone globular domain and has been associated with transcriptional activity, but also with transcriptionally inert genomic regions22. The modification is evolutionarily conserved since it has been observed in yeast, calf thymus, chicken, and human23. H3K79 mono, di, and trimethylation (H3K79me1, me2, me3) are catalyzed by the histone methyltransferases DOT1L24,25 and the Nuclear SET Domain-Containing Protein 2 (Nsd2)26. DOT1L is implicated in proliferation, DNA repair, and cellular reprograming27. Loss of Dot1l in mice leads to a prenatal death around the developmental stage E10.528,29. During heart development and in myocardiocyte differentiation, DOT1L is essential for gene expression regulation30. In the central nervous system, DOT1L function might be implicated in neural tube development31, it is involved in suppressing Tbr1-expression during forebrain development32, and may function in the regulation of ER-stress response genes33. The context-dependent activating or repressing action of H3K79me, especially with in vivo situations like the development of the central nervous system, is to date only partially understood32. Since H3K79 methylation is located in the globular domain of histone 3, it is sterically less accessible in comparison to modifications on the flexible histone tails23. To understand the function of H3K79 methylation, reliable and reproducible analysis methods to determine its location and genomic environment are needed. In this methods paper, we present isolation methods of different neural progenitors (CPCs for the cortex and CGNPs for the cerebellum), effective DOT1L inhibitor treatment, and a ChIP method to analyze H3K79 methylation via qPCR or sequencing at different time points during cortical and cerebellar development. For an overview of the protocol and its possibilities, see Figure 1.

<table><tbody><tr><td>Anti-GAPDH</td><td>Abcam</td><td>ab8245</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:5000</td></tr><tr><td>Anti-H3</td><td>Abcam</td><td>ab1791</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:3000</td></tr><tr><td>Anti-H3K79me2</td><td>Diagenode</td><td>pAb-051-050</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; ChIP antibody</td></tr><tr><td>Anti-H3K79me2</td><td>Abcam</td><td>ab-051-050</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:1000</td></tr><tr><td>Anti-rabbit-IgG</td><td>Diagenode</td><td>C15410206</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; ChIP Ctrl antibody</td></tr><tr><td>Anti-Tubulin alpha</td><td>Abcam</td><td>ab108629</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Antibody&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Immunoblot dilution 1:3000</td></tr><tr><td>Apo-Transferrin (1 mg/ml)</td><td>Sigma-Aldrich</td><td>T1147</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>B27 Supplement (50x)</td><td>Life Technologies</td><td>17504044</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Bioanalyzer</td><td>Agilent technologies</td><td>G2940CA</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For analysis of sheared chromatin</td></tr><tr><td>Bioruptor NextGen</td><td>Diagenode</td><td>B01020001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Ultrasonicator</td></tr><tr><td>Boric acid pH 8.4</td><td>Sigma Aldrich</td><td>B6768</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>CFX Connect RT PCR Detection System</td><td>Bio-Rad</td><td>1855201</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Detection system for qPCR</td></tr><tr><td>DMEM-F12</td><td>Life Technologies</td><td>11320-033</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGM</td></tr><tr><td>Dynabeads Protein A</td><td>Invitrogen</td><td>10001D</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Magnetic beads, for ChIP</td></tr><tr><td>EPZ-5676</td><td>Selleckchem</td><td>S7062</td><td>&lt;strong&gt;Category:&lt;/strong&gt; DOT1L inhibition&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For DOT1L inhibition in cell culture</td></tr><tr><td>Ethylenediamine tetraacetic acid</td><td>SERVA</td><td>39760.01</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; EDTA</td></tr><tr><td>Fetal Bovine Serum 10% (v/v)</td><td>Gibco</td><td>10082147</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC isolation and CGM</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Glutathione (1.25 mg/ml)</td><td>Sigma-Aldrich</td><td>G4251</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>3187</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For cell fixation</td></tr><tr><td>GoTaq mastermix</td><td>Promega</td><td>A6002</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; DNA polymerase master mix for qPCR</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Life Technologies</td><td>14025-100</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; HBSS</td></tr><tr><td>L-glutamine (200 mM)</td><td>Life Technologies</td><td>25030081</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>Lithium chloride</td><td>Sigma-Aldrich</td><td>L4408</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; LiCl</td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502048</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGM</td></tr><tr><td>NanoDrop 3300</td><td>Thermo Fisher</td><td>3300</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Fluorospectrometer for DNA quantification</td></tr><tr><td>NEB Next Ultra DNA Library Prep Kit for Illumina</td><td>NEB</td><td>E7645S</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Kit for Library preparation</td></tr><tr><td>NEBNext Multiplex Oligos for Illumina</td><td>NEB</td><td>E7335</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Oligos for Library preparation</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>NP-40 Alternative</td><td>Calbiochem</td><td>492016</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP buffer</td></tr><tr><td>Paraformaldehyde</td><td>Carl Roth</td><td>335</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PFA, for cell fixation</td></tr><tr><td>Penicillin-Streptomycin-Neomycin 1% (v/v)</td><td>Life Technologies</td><td>15640055</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PSN, for CCM and CGM</td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010023</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; PBS, for CPC isolation</td></tr><tr><td>PicoGreen Kit</td><td>Thermo Fisher</td><td>P11496</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP Analysis&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Visualizing dye for DNA quantification</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Poly-L-ornithine hydrobromide</td><td>Sigma Aldrich</td><td>P3655</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC culturing</td></tr><tr><td>Potassium chloride</td><td>Thermo Fisher</td><td>AM9640G</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; KCl, for CGM</td></tr><tr><td>Protease inhibitor</td><td>Roche</td><td>4693159001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>3115879001</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Qiagen MinElute</td><td>Qiagen</td><td>28004</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; Kit for DNA purification</td></tr><tr><td>RNAse</td><td>Sigma-Aldrich</td><td>R6513</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>SGC0946</td><td>Selleckchem</td><td>S7079</td><td>&lt;strong&gt;Category:&lt;/strong&gt; DOT1L inhibition&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For DOT1L inhibition in cell culture</td></tr><tr><td>Sodium bicarbonate</td><td>Carl Roth</td><td>8551.1</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; for Elution buffer</td></tr><tr><td>Sodium chloride</td><td>Carl Roth</td><td>9265</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; NaCl, for ChIP buffer</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>30970</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP</td></tr><tr><td>Sodium dodecylsulfate</td><td>Carl Roth</td><td>183</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; SDS, for ChIP</td></tr><tr><td>Sonic hedgehock (SHH)</td><td>Sigma-Aldrich</td><td>SRP6004</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CGNP isolation</td></tr><tr><td>Superoxide dismutase (1mg/ml)</td><td>Sigma-Aldrich</td><td>S7571</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CCM</td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Carl Roth</td><td>9090</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; TRIS, for ChIP buffer</td></tr><tr><td>Triton X-100</td><td>Carl Roth</td><td>X100</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For ChIP buffer</td></tr><tr><td>Trypsin-EDTA 0,05% (w/v)</td><td>Sigma Aldrich</td><td>59417C</td><td>&lt;strong&gt;Category:&lt;/strong&gt; Cell culture&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For CPC isolation</td></tr><tr><td>Tween20</td><td>Carl Roth</td><td>28320</td><td>&lt;strong&gt;Category:&lt;/strong&gt; ChIP&lt;br/&gt; &lt;strong&gt;Abbreviation/Comment:&lt;/strong&gt; For bead preparation</td></tr><tr><td>Other Lab devices: Neubauer counting chamber, Incubator, Rotator, Shaker, Disection set, Water bath</td><td></td><td></td><td></td></tr><tr><td>CCM: Cortical cell medium</td><td></td><td></td><td></td></tr><tr><td>CGM: CGNP cell culture medium</td><td></td><td></td><td></td></tr></tbody></table>

isolation and cultivation of neural progenitors followed by chromatin-immunoprecipitation of histone 3 lysine 79 dimethylation mark

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

General scheme of neural progenitor isolation, cultivation, H3K79me2 ChIP and ChIP analysis methods: Figure 1 shows a flowchart to perform H3K79me2 ChIP of cortical progenitor cells at different time points during embryonic brain development or of cerebellar granular neuron progenitors in postnatal stages. As a first step, the brain has to be isolated and the telencephalon (between E11.5 and E14.5) or the cerebellum (P5-P7) has to be retrieve...

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

isolation-cultivation-neural-progenitors-followed-chromatin

Research

JoVE Journal

Neuroscience

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture.  Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Source: Tagliafierro, L., et al. Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models. J. Vis. Exp. (2019)This video demonstrates the lentiviral transduction of neural progenitor cells (NPCs) for targeted epigenetic modification of alpha-synuclein, a key protein in Parkinson’s disease. The protocol enables methyltransferase-mediated DNA methylation, reducing SNCA expression for studying gene regulation...

Lentiviral Transduction of Neural Progenitor Cells for Epigenetic Regulation of Alpha-Synuclein Expression

This video demonstrates a technique for isolating induced neural progenitor cells (iNPCs) derived from adult human fibroblasts. It outlines the steps involved in isolating the iNPC colonies and culturing them in laminin-coated multi-well plate wells in the presence of suitable culture media that facilitate the continued proliferation and expansion of the iNPC population.

Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup's cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres. J. Vis. Exp.  (2018).This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Native Chromatin Immunoprecipitation of Neurosphere Cells

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Learn to isolate and culture neural progenitor cells from cerebellar tissue for epigenetic analysis using ChIP. This method enables study of H3K79me2 histone modifications during brain development.

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark (Video) | JoVE Journal

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

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