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Neuroscience

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

Published: April 16th, 2021

DOI:

10.3791/62405

1Department of Pathology, Icahn School of Medicine at Mount Sinai, 2Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 3Institutes of Brian Science, Fudan University, 4Department of Psychiatry, Icahn School of Medicine at Mount Sinai

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 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 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 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.

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 th....

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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 "human research" designation by ISMMS's IRB (HS#14-01007). 

1. Buffer prepara.......

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Nuclei were collected from fresh (unfixed) snap-frozen 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

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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.......

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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'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.).

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

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