Gene expression profile at the single cell level of CD45+ CD11b+ cells isolated from the brain and bone marrow of mice either untreated or conditioned and transplanted with total bone marrow.
We used single-cell RNA sequencing (scRNA-seq) to analyze the diversity of bone marrow-derived CD45+CD11b+ microglia-like cells (MGLCs) engrafted in the brain of recipient mice that were conditioned using Busulfan and PLX3397 and transplanted with total bone marrow. We compared the gene expression of MGLCs to that of developmentally-derived CD45+ CD11b+ microglia/myeloid cells isolated from the brain of recipient mice (host microglia) and untreated mice (naive microglia). We also compared the gene expression of MGLCs to that of transplant-derived CD45+ CD11b+ cells engrafted in the bone marrow (abbreviated as BM-CD11b+) Overall design: Brain CD45+ CD11b+ cells were isolated by Fluorescence-activated cell sorting (FACS) based on the co-expression of the CD45+ and CD11b+ surface markers and the expression of the Green Fluorescent Protein (GFP). Four CD45+ CD11b+ cell samples were isolated by FACS and used for the scRNA-seq. Cells isolated from C57BL/6 mice (Jax strain #000664) conditioned with Busulfan (100 mg/kg) and PLX3397 (600 mg/kg) and transplanted with total bone marrow harvested from adult sex-matched C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ mice (Jax strain #006567) were: 1)  Sample A or MGLCs: GFP+ CD45+ CD11b+ MGLCs, isolated from the brain; 2)  Sample B or host conditioned MG or, host MG: GFP- CD45+ CD11b+ cells,  isolated from the brain;  3) Sample D or BM-CD11b+: GFP+ CD45+ CD11b+ cells, isolated from the bone marrow; the time point of the analysis is 4 months after transplant. Cells isolated from untreated age-matched donor mice [C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ, (Jax strain #006567] were: 4) Sample C or naive MG: GFP+ CD45+ CD11b+ cells isolated from the brain.