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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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

Discussion

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.......

Acknowledgements

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.).

....

Materials

NameCompanyCatalog NumberComments
10x PBS pH 7.2Invitrogen70013073
ANTI-NEUN ANTIBODY CLONE A60MilliporeMAB377A5MImouse anti-NeuN conjugated to a fluorescent compound AF555 (excitation, 553 nm; emission, 568 nm)
ANTI-OLIG2 ANTIBODY CLONE 211MilliporeMABN50A4MImouse anti-OLIG2 conjugated to a fluorescent compound AF488 (excitation, 499 nm; emission, 520 nm)
Bovine Serum AlbuminFisherBP9704-100
Bright-Line Counting ChamberHausser Scientific3110V
Calcium Chloride AnhydrousFisherC614-3
Cell Strainers, 40 µMSP Scienceware136800040
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)InvitrogenD1306
DL-DithiothreitolSigma43815-1G
DNA Library KitIllumina, NexteraFC-121–1030
DNAse IWorthingtonLS002139
Dounce Tissue GrinderWHEATON357542
FACS SorterBD BiosciencesBD FACSAria III
Magnesium Acetate TetrahydrateFisherM13-500
PAX6 (PAX6/496) - 100 TESTSNovusNBP234705J
RNA Clean & ConcentratorZymo ResearchR1013
RNaseZap RNase Decontamination SolutionInvitrogenAM9780
SMARTer Stranded Total RNA-Seq Kit Pico Input MammalianClontech Laboratories635005Fragmentation time of 2.5 minutes, as recommended for low RIN RNA values.
Sucrose, crystal certified, ACS, 500 mgFisherS5500
SW 41 Ti Swinging-Bucket RotorBeckman Coulter331362
Tris-HCl, 1M Solution, pH 8.0, Molecular Biology Grade, UltrapureThermo ScientificJ22638AE
TritonX-100FisherBP151-500non-ionic surfactant in lysis buffer
TRIzol LS ReagentInvitrogen10296028
TRIzol ReagentInvitrogen15596026reagent for isolation of RNA
Trypan Blue Solution, 0.4%Gibco15250061
UltracentrifugeBeckman Coulter Optima XE-100A94516
Ultracentrifuge tubes PP 9/16 X 3-1/2Beckman Coulter331372
UltraPure Distilled Water (RNAse-, DNAse-free)Invitrogen10977023referred to as distilled water
Ultrapure EDTALife Technologies15576-028

References

  1. Mitchell, A., Roussos, P., Peter, C., Tsankova, N., Akbarian, S. The future of neuroepigenetics in the human brain. Progress in Molecular Biology and Translational Science. 128, 199-228 (2014).
  2. Jiang, Y., Matevossian, A., Huang, H. S., Straubhaar, J., Akbarian, S.

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