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.
