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 mouse hematopoietic stem and progenitor cells (HSPCs) expanded in culture.
We used single-cell RNA sequencing (scRNA-seq) to analyze the diversity of HSPC-derived CD45+CD11b+ microglia-like cells (MGLCs) engrafted in the brain of recipient mice that were conditioned using Busulfan and PLX3397 and transplanted intravenous with Lineage negative KIT+ SCA1+ mouse HSPCs. The HSPCs were isolated from adult C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ homozygous mice. 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 (125 mg/kg) and PLX3397 (600 mg/kg) and transplanted with HSPCs 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 9 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. Cell Hashing, using oligo-tagged antibodies directed to mouse CD45 (clone 30-F11) and mouse MHCI (clone M1/42), was used to uniquely label cells from 3 distinct mice/sample (multiplexing).  Details on the cell hashing and hashtag oligo (HTO) are included in the "extract protocol" section below.