Neural Stem Cell Relay from B1 to B2 cells in the adult mouse Ventricular-Subventricular Zone
Neurogenesis and gliogenesis continue in the Ventricular-Subventricular Zone (V-SVZ) of the adult rodent brain. B1 cells are radial glia-derived astroglial cells that function as primary progenitors or neural stem cells (NSCs) in the V-SVZ.  B1 cells, which have an apical contact with the ventricle, decline during early postnatal life, yet neurogenesis continues into adulthood. We found that a second population of V-SVZ astroglial cells (B2 cells), that do not contact the ventricle, function as NSCs in the adult brain. B2 cell numbers increase postnatally, remain constant in 12-month-old mice, and decrease by 18 months. Transcriptomic analysis revealed differences between B1 and B2 cells and shows that like B1 cells, B2 cells can be quiescent or activated.  Transplantation and lineage tracing of B2 cells demonstrate their function as primary progenitors for adult neurogenesis. This study reveals that NSC function is relayed from B1 to B2 progenitors to maintain adult neurogenesis. Overall design: 10x Technology single cell sample preparation and multiplexing. Samples from mice injected with Ad:Cre:GFP were prepared as following:  15-24 hours after Ad:Cre:GFP intraventricular injections, mice (Experiment 1(ACSAAB01): n=14, P27-30, Experiment 2 (ACSAAB02): n=12, P27-p30) received intraperitoneal administration of 2.5% Avertin followed by decapitation. Brains were extracted and 0.5 mm slices were obtained with a mouse brain slicer (Steel Brain Matrix - Coronal 0.5mm, Alto). The V-SVZ lateral wall and dorsal wedge were microdissected and considered separate samples. To analyze the transcriptomic profile of aged mice we also microdissected the V-SVZ of two non-infected hGFAP:GFP 1 year old mice. The wedge region was not included. All V-SVZ micro-dissections were performed in L-15 medium on ice and the tissue was transferred to Papain-EBSS (LK003150, Worthington). Tissue was digested for 30 min at 37ºC in a thermomixer at 900 RPM. Mechanical dissociation with a P1000 pipette tip (20 seconds), then with a  fire-polished pasteur pipette for 3 min.  Cells were centrifuged for 5 min, 300 RCF at room temp, and the pellet was resuspended with DNAase/ovomucoid inhibitor according to the manufacturer's protocol (Worthington). Cells were incubated in red blood cell lysis buffer (420301, Biolegend) 3-4 min at 4ºC. For MULTI-seq barcoding, cells were suspended with Anchor:Barcode solution for 5 minutes at 4ºC. A Co-Anchor solution was added and incubated for 5 min87. Samples were combined and filtered with a FlowMi 40µm filter (BAH136800040-50EA, Sigma). To remove myelin, the cell suspension was incubated with Myelin Removal Beads (130-096-733, Miltenyi Biotec) (6µl/brain) for 15 min at 2-8°C. Cells were washed with 0.5% BSA-PBS and transferred to MACS columns. The effluent was collected, and the cell density was counted. Cells were loaded into a 10x Genomics Chromium Single Cell Controller. We used the 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit v3.1 to generate cDNA libraries for sequencing according to the manufacturer's protocols. MULTI-Seq barcode libraries were prepared according to MULTI-Seq (McGinnis et al. 2019)  protocols. We confirmed that isolated cells from wedge dissections did not contain apical cells (GFP+ and/or  TdTomato+ cells) under an epifluorescence microscope. Libraries were sequenced in an Illumina NovaSeq 6000. Alignment was performed on STAR v2.7.11 and included both intronic and exonic mappings (--soloFeatures Gene GeneFull Velocyto). We used an optimized mouse reference genome 88 edited to include GFP and Cre genes. We also included a WPRE sequence to serve as a proxy for TdTomato expression.