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

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.......

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.......

Materials

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