We like to thank members in Pathology and Neurosurgery at the Icahn School of Medicine at Mount Sinai for help with the procurement of de-identified brain tissue and ISMMS&#39;s Flow Cytometry CORE for expert advice. The study was partially funded by NIH RF1DA048810, R01NS106229, R03NS101581 (to N.M.T.), and R61DA048207 (to S.A.).

NOTE: The Program for the Protection of Human Subjects at the Icahn School of Medicine at Mount Sinai (ISMMS) and its Institutional Review Board (IRB) assures the ethical conduct of research and compliance with federal, state, and institutional regulations. In this study, all postmortem specimens used were de-identified, obtained under appropriate consent through the biorepository, and were exempt from &#34;human research&#34; designation by ISMMS&#39;s IRB (HS#14-01007).&#160;
1. Buffer prepara...

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N., Mi, D., Mason, J. O., Price, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cerebral+cortical+neurogenesis+by+the+Pax6+transcription+factor.">Regulation of cerebral cortical neurogenesis by the Pax6 transcription factor.</a> Frontiers in Cellular Neuroscience. 9, 70 (2015).</li><li>Sakurai, K., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurogenesis-controlling+factor,+Pax6,+inhibits+proliferation+and+promotes+maturation+in+murine+astrocytes.">The neurogenesis-controlling factor, Pax6, inhibits proliferation and promotes maturation in murine astrocytes.</a> Journal of Neuroscience. 28 (18), 4604-4612 (2008).</li><li>Zhong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+development+of+the+human+hippocampus.">Decoding the development of the human hippocampus.</a> Nature. 577 (7791), 531-536 (2020).</li><li>Pollen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identity+of+human+outer+radial+glia+during+cortical+development.">Molecular identity of human outer radial glia during cortical development.</a> Cell. 163 (1), 55-67 (2015).</li><li>Cvekl, A., Callaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX6:+25th+anniversary+and+more+to+learn.">PAX6: 25th anniversary and more to learn.</a> Experimental Eye Research. 156, 10-21 (2017).</li><li>Goc, J., Liu, J. Y., Sisodiya, S. M., Thom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatiotemporal+study+of+gliosis+in+relation+to+depth+electrode+tracks+in+drug-resistant+epilepsy.">A spatiotemporal study of gliosis in relation to depth electrode tracks in drug-resistant epilepsy.</a> European Journal of Neuroscience. 39 (12), 2151-2162 (2014).</li><li>Monoranu, C. 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The authors have nothing to disclose.

Experimental design following the outlined protocol should be finalized after considering several biological and technical factors. Starting tissue samples are fresh-frozen, without having been fixed, and preferably have a short postmortem collection interval to maximize nuclei recovery. Based on experience, a PMI of up to 24 h allows for adequate nuclei recovery; however, a PMI of 12 h or less is preferable to optimize intact nuclei recovery. Additional factors apart from PMI, including temperature of body storage and p...

The molecular complexity of human astrocytes remains poorly defined in primary tissue, requiring better tools for their isolation and characterization at high resolution, both in health and disease. Separation of intact human neurons and glia from their niche has proven difficult due to limited access of fresh brain tissue samples, the heavily interconnected nature of glial and neuronal processes, and inevitable cellular activation during processing, all of which limit the molecular characterization of these cell types ex vivo1. Fluorescence-activated nuclei sorting (FANS) has emerged as an alternative to live-cell sorting, enabling the dissociation and immunotagging of nuclei populations from frozen tissue. In the past decade, FANS has become widely used for isolating and molecularly characterizing human neuronal (NeuN+) nuclei populations in a variety of brain specimens and anatomical regions1,2,3,4.
However, similar methods for isolating specific glial nuclei subpopulations from human cortex have been limited, leading to a relative lack of sophistication in the understanding of astrocyte complexity in both normal and diseased tissues. To this end, a previously published protocol was adapted for isolating human neuronal populations using FANS4, and a method was validated to enrich for astrocytes (and for oligodendroglial progenitors) using a triple-antibody combination, capturing astrocytes in both resting and reactive conditions5. To specifically enrich for astrocytes in the NeuN- fraction, antibodies were used against one of two transcription factors known to be differentially expressed across astrocyte populations, PAX6 or SRY-box transcription factor 9&#160;(SOX9)6,7. PAX6 is highly expressed during early fetal development within radial glia-like progenitors in germinal zones and contributes toward both neurogenesis and gliogenesis8,9,10,11 as well as to retinal neuronal specification12. In the adult, PAX6 is differentially overexpressed in resting human astrocytes6 and shows protein co-expression with glial fibrillary acidic protein (GFAP) in human epilepsy tissue astrocytes13.
This protocol describes the simultaneous isolation of neocortical neuronal and astrocyte-enriched nuclei populations by FANS. Fresh (unfixed) snap-frozen (i.e., fresh-frozen)&#160;postmortem tissue collected from adult cortex is first mechanically and chemically dissociated. After lysis and ultracentrifugation in a sucrose gradient, the cytoplasmic and extracellular components are discarded while the nuclei are retained. Nuclei are then labeled with fluorescently conjugated nuclear antibodies corresponding to the desired target lineages and sorted using FANS. Following this approach, enrichment of astrocytes is demonstrated in the collected PAX6+NeuN- populations, validated both by a targeted qPCR panel as well as by downstream nuclear RNA sequencing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x PBS pH 7.2</td>
			<td>Invitrogen</td>
			<td>70013073</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-NEUN ANTIBODY CLONE A60</td>
			<td>Millipore</td>
			<td>MAB377A5MI</td>
			<td>mouse anti-NeuN conjugated to a fluorescent compound AF555 (excitation, 553 nm; emission, 568 nm)</td>
		</tr>
		<tr>
			<td>ANTI-OLIG2 ANTIBODY CLONE 211</td>
			<td>Millipore</td>
			<td>MABN50A4MI</td>
			<td>mouse anti-OLIG2 conjugated to a fluorescent compound AF488 (excitation, 499 nm; emission, 520 nm)</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP9704-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Counting Chamber</td>
			<td>Hausser Scientific</td>
			<td>3110V</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Anhydrous</td>
			<td>Fisher</td>
			<td>C614-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers, 40 &#181;M</td>
			<td>SP Scienceware</td>
			<td>136800040</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma</td>
			<td>43815-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Library Kit</td>
			<td>Illumina, Nextera</td>
			<td>FC-121&#8211;1030</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder</td>
			<td>WHEATON</td>
			<td>357542</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>BD FACSAria III</td>
		</tr>
		<tr>
			<td>Magnesium Acetate Tetrahydrate</td>
			<td>Fisher</td>
			<td>M13-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX6 (PAX6/496) - 100 TESTS</td>
			<td>Novus</td>
			<td>NBP234705J</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>R1013</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTer Stranded Total RNA-Seq Kit Pico Input Mammalian</td>
			<td>Clontech Laboratories</td>
			<td>635005</td>
			<td>Fragmentation time of 2.5 minutes, as recommended for low RIN RNA values.</td>
		</tr>
		<tr>
			<td>Sucrose, crystal certified, ACS, 500 mg</td>
			<td>Fisher</td>
			<td>S5500</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, 1M Solution, pH 8.0, Molecular Biology Grade, Ultrapure</td>
			<td>Thermo Scientific</td>
			<td>J22638AE</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td>non-ionic surfactant in lysis buffer</td>
		</tr>
		<tr>
			<td>TRIzol LS Reagent</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>reagent for isolation of RNA</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter Optima XE-100</td>
			<td>A94516</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes PP 9/16 X 3-1/2</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water (RNAse-, DNAse-free)</td>
			<td>Invitrogen</td>
			<td>10977023</td>
			<td>referred to as distilled water</td>
		</tr>
		<tr>
			<td>Ultrapure EDTA</td>
			<td>Life Technologies</td>
			<td>15576-028</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of adult human astrocyte populations from fresh-frozen cortex using fluorescence-activated nuclei sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

Nuclei were collected from fresh (unfixed) snap-frozen&#160;temporal neocortex tissue with a postmortem collection time of 12 h. After tissue dissociation into nuclei suspension, samples were incubated with antibodies against NeuN, PAX6, and OLIG2, and sorted according to the gating shown in Figure 1 and Figure 2. Nuclei were collected from NeuN+, PAX6+NeuN-, and OLIG2+NeuN- sorted populations (Figure 1E,F and <stro...

Watch this Scientific Journal Video about Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting at JoVE.com

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

This protocol can help scientists who are interested in studying the molecular function of human astrocytes in their native niche. The main advantage of this technique is that it allows for the isolation and enrichment of a specific cell type, adult astrocytes from bulk human brain tissue specimens using fluorescence-activated nuclei sorting. This methodology can be applied to isolating other cell types from the human neocortex, such as neurons and oligodendrocyte progenitor cells using other appropriate fluorescent-conjugated antibodies.Begin dissecting approximately 200 to 400 milligrams of tissue from a fresh frozen human adult cortex tissue sample. Place the tissue in a seven-milliliter glass tissue douncer containing four milliliters of ice-cold lysis buffer on ice and grind the tissue approximately 50 times. Transfer the homogenate to a 12-milliliter ultracentrifuge polypropylene tube.Use a five-milliliter pipette to add 6.5 milliliters of ice-cold sucrose buffer to the bottom of the ultracentrifuge tube without disturbing the interface between the sucrose and the tissue homogenate. To collect the nuclei, ultracentrifuge the lysate for one hour at 101, 814 times G and carefully aspirate the supernatant and debris without disturbing the pellet. Add 0.1%BSA in PBS to the ultracentrifuge tube for a 10-minute incubation on ice before resuspending the nuclei pellet.Then, use trypan blue to visualize the intact nuclei under a light microscope and to ensure that the concentration is above 10 to the fifth nuclei per milliliter For fluorescence-activated sorting of the isolated nuclei, add approximately 20 microliters of the resuspended sample to an antibody solution containing Alexa fluor 555 conjugated mouse anti-NeuN antibody. Add approximately 20 microliters of the resuspended sample to an antibody solution containing APC-conjugated mouse anti-PAX6 antibody. After a one-hour incubation at four degrees Celsius with shaking, protected from light, add DAPI at a 1:1000 ratio to all of the samples and controls.To include nuclei of the appropriate size and to exclude red blood cells and debris, use a control tube to gate the cells by their forward and side scatter areas. To select the singlet nuclei population, gate by forward scatter area versus forward scatter width or height and side scatter area versus side scatter width or height. Gate by DAPI to include only the intact nuclei singlets and to exclude any debris and doublets.After gating according to any additional fluorescence controls, run the DAPI-only control sample. Run the NeuN Alexa fluor 555-only control to determine the cutoff for the NeuN-positive staining in the Alexa fluor 555 channel. Run the PAX6 APC-only control to determine the cutoff for the PAX6-positive staining in the APC channel.After all of the controls have been run, gate the NeuN-positive cells to collect the neurons and gate the PAX6-positive cells with the NeuN-negative population to collect the astrocytes. Gate and collect any additional glial populations of interest from the NeuN-negative PAX6-negative population. For bulk single-nucleus RNA sequencing, after collecting 50, 000 to 500, 000 nuclei in 0.04%BSA-supplemented PBS, add two milliliters of sucrose solution, 50 microliters of one-molar calcium chloride, and 30 microliters of one-molar magnesium acetate to the sample.Bring the final volume of the suspension up to 10 milliliters with PBS. Mix the tube contents by inversion and incubate the reaction on ice for 15 minutes. Pellet the nuclei by centrifugation and resuspend the nuclei in one-milliliter of RNA extracting reagent.Then, vortex the tube, freeze the sample on dry ice, and store the nuclei at minus 80 degrees Celsius. For bulk single-nucleus transposase-acceptable chromatin sequencing, collect 50, 000 to 75, 000 nuclei into 0.04%BSA-supplemented PBS in a microcentrifuge tube coated with 5%BSA and freeze the nuclei until their downstream analysis. Neuronal, astrocyte, and oligodendrocyte progenitor nuclei populations can be sorted from a fresh frozen postmortem human temporal neocortex sample as demonstrated.Single-nucleus RNA sequencing studies further prioritized PAX6 as a top differentially-expressed nuclear transcription factor across both protoplasmic and fibrous adult astrocytes subpopulations. After sorting, cell type-specific transcriptome alterations in the primary pathological epilepsy neocortex can be characterized. In this analysis, transcriptomic analyses confirmed that the PAX6-positive NeuN-negative populations were robustly enriched for panastrocyte markers and depleted for neuronal markers.Immunofluorescence staining can be used to confirm the co-localization of PAX6 with glial fibrillary acidic protein in human cortical astrocytes. Shorter postmortem intervals are associated with a greater intact nuclei recovery from fresh tissue samples. A high-yield of intact nuclei can be recovered from frozen tissue up to 24 hours postmortem, but at 30 hours, very few intact nuclei can be recovered.The most important thing to remember is to get the PAX6-positive population from the NeuN-negative population, because there are neurons that also express PAX6, and we want to exclude those. This technique also allowed the study of astrocytes in epilepsy surgical brain specimens, further elucidating the molecular dysregulation of human astrocytes in the context of a specific pathological niche.

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3.2K Görüntüleme.  Icahn School of Medicine at Mount Sinai. Bu protokol, insan astrositlerinin moleküler fonksiyonlarını doğal nişlerinde incelemek isteyen bilim insanlarına yardımcı olabilir. Bu tekniğin temel avantajı, floresan ile aktive olmuş çekirdekleri sıralamayı kullanarak toplu insan beyin dokusu örneklerinden belirli bir hücre tipinin, yetişkin astrositlerin izole edilmesine ve zenginleştirilmesine izin vermesidir. Bu metodoloji, diğer uygun floresan konjuge antikorları kullanarak nöronlar ve oligodendrosit progenitör h...