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.. 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.

本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，分析经白消安（Busulfan）与PLX3397预处理、接受全骨髓移植的受体小鼠脑内植入的骨髓源性CD45+CD11b+小胶质细胞样细胞（microglia-like cells, MGLCs）的多样性。本研究将MGLCs的基因表达与三类对照细胞进行对比：一是从受体小鼠脑组织中分离得到的发育源性CD45+CD11b+小胶质细胞/髓系细胞（宿主小胶质细胞），以及从未处理小鼠脑组织中分离得到的对应细胞（初始小胶质细胞/naive microglia）；二是植入受体小鼠骨髓的移植源性CD45+CD11b+细胞（缩写为BM-CD11b+）。
整体实验设计如下：通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS），基于CD45+与CD11b+表面标志物的共表达以及绿色荧光蛋白（Green Fluorescent Protein, GFP）的表达，分离目标CD45+CD11b+细胞，最终获得4份细胞样本用于scRNA-seq分析。
本次实验的细胞样本分为两类来源：
1. 来源于经白消安（100 mg/kg）与PLX3397（600 mg/kg）预处理、接受成年同性别C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ小鼠（Jax品系#006567）全骨髓移植的C57BL/6小鼠（Jax品系#000664），包含3份样本：
- 样本A（MGLCs组）：从受体小鼠脑组织中分离得到的GFP+CD45+CD11b+ MGLCs；
- 样本B（宿主小胶质细胞组/host MG组）：从受体小鼠脑组织中分离得到的GFP-CD45+CD11b+细胞；
- 样本D（BM-CD11b+组）：从受体小鼠骨髓中分离得到的GFP+CD45+CD11b+细胞；本研究的分析时间点为移植后4个月。
2. 来源于未处理的同年龄供体小鼠（C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ，Jax品系#006567），包含1份样本：
- 样本C（初始小胶质细胞组/naive MG组）：从供体小鼠脑组织中分离得到的GFP+CD45+CD11b+细胞。