RNAseq for cultured Control and TgAPPsweOCN BMSCs (sorted Ai9+ Osteoblast-linage cells)

Purpose: To investigate if and how APPswe in OCN-Cre+ OB-lineage cells gives rise to the brain and behavior phenotypes, we purified OCN-Cre+ BMSCs (marked by tdTomato+, believed to be OB progenitors) from both the 6-MO control (OCN-Cre; Ai9) and TgAPPsweOCN; Ai9 mice using fluorescence-activated cell sorting (FACS) and then subjected them to RNA-seq analysis Methods: (1)the whole bone marrow cells of 6-month-old OCn-Cre;Ai9 andTgAPPswe;OCn-Cre;Ai9 flushed out from long bones of mice with DMEM were filtered through a 70-mm filter mesh, washed, re-suspended, and then plated in 100-mm dishes with growth medium (DMEM plus 10% FBS), which were incubated at 37oC with 5% CO2. The non-adherent cells were removed 72 hours after changing the medium. The attached bone marrow cells were cultured with the growth medium for 7 days. These cells were then resuspended and plated at culture dishes and cultured for another 3-6 days with the same growth medium. These cells, so called BMSCs. (2)Then cell media were removed from culture dishes and cells were rinsed with PBS. Trypsin solution was added to incubate at 37°C for 2 min. The detached adherent cells were centrifuged, and the pellet cells were washed with 1 ml cold PBS, and finally resuspended in 0.5 ml PBS with 1% FBS for flow cytometry analysis. Flow cytometric analysis was performed by use of a flow cytometer in CWRU core facility. Acquisition and analysis were performed by using FACSDiva 8.0.1 software (BD).(3)Total RNAs were extracted from purified Td+ OB-progenitor cells from OCN-Cre; Ai9 and TgAPPsweOCN; Ai9 mice by flow cytometer. RNA Integrity Number (RIN) was accessed for every sample, and the samples were considered qualified with RIN>?2. These RNA samples were then subjected to RNA-seq analyses by BGI America (Cambridge, MA) using the DNBseq platform. Firstly, we removed the reads mapped to rRNA and obtained the raw data with 52.47 Mb reads. After filtering low-quality, adaptor-polluted and high content of unknown base reads in the sequencing reads, 51.9 Mb clean reads were obtained per sample on average. Then clean reads were mapped to reference genome using HISAT2. On average 92.91% reads were mapped and the uniformity of the mapping result for each sample suggests that the samples were comparable. Comparisons to RNAseq were normalized to fpkm Results: 917 up- and 1825 down-regulated genes were identified in APPswe+ OB progenitors. Among these genes, 154 up- and 269 down-regulated genes encode secreted proteins. Interestingly, go analysis showed that most up-regulated genes are involved in inflammatory response, cytokine production, and cytokine/chemokine-mediated signaling pathways; and most down-regulated genes are implicated in cell cycle, cell proliferation, and bone mineralization. Conclusions: Increased cytokines and chemokines in APPswe+ OB-progenitors, indicating that APPswe+ OB progenitors may undergo cellular senescence. Overall design: cultured BMSCs mRNA profiles of 6-month-old OCn-Cre;Ai9 andTgAPPswe;OCn-Cre;Ai9 mice

研究目的：为探究OCN-Cre阳性成骨细胞谱系细胞中的APPswe突变是否以及如何引发大脑与行为表型，我们通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS），从6月龄对照小鼠（OCN-Cre; Ai9）及TgAPPsweOCN; Ai9小鼠中纯化得到OCN-Cre阳性骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells, BMSCs，该细胞经tdTomato阳性标记，被认为是成骨细胞前体），随后对其进行RNA测序（RNA-seq）分析。
实验方法：
(1) 从6月龄OCN-Cre;Ai9及TgAPPsweOCN;OCN-Cre;Ai9小鼠长骨中冲出的骨髓全细胞，经达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）重悬后，通过70微米滤膜过滤，洗涤并重悬，接种于含生长培养基（DMEM添加10%胎牛血清（Fetal Bovine Serum, FBS））的100mm培养皿中，于37℃、5%二氧化碳培养箱中孵育。换液72小时后移除非贴壁细胞，贴壁骨髓细胞继续用生长培养基培养7天。随后将细胞重悬并接种至培养皿，用相同生长培养基再培养3-6天，所得细胞即为骨髓间充质干细胞。
(2) 随后移除培养皿中的培养基，用磷酸盐缓冲液（Phosphate Buffered Saline, PBS）冲洗细胞。加入胰蛋白酶（Trypsin）溶液，于37℃孵育2分钟。将脱落的贴壁细胞离心，沉淀用1mL预冷PBS洗涤，最终重悬于含1% FBS的0.5mL PBS中用于流式细胞术分析。流式细胞术分析在凯斯西储大学核心设施的流式细胞仪上完成，数据获取与分析采用FACSDiva 8.0.1软件（BD公司）。
(3) 通过流式细胞术从OCN-Cre;Ai9及TgAPPsweOCN;Ai9小鼠中纯化的tdTomato阳性成骨细胞前体中提取总RNA。对每一份样本检测RNA完整性指数（RNA Integrity Number, RIN），RIN值大于2的样本判定为合格。随后将合格的RNA样本交由美国华大基因（BGI America，马萨诸塞州剑桥市）采用DNBseq平台进行RNA-seq分析。首先，移除比对至核糖体RNA（rRNA）的序列，获得含52.47 Mb读段的原始数据；对测序读段进行过滤，去除低质量、接头污染及高未知碱基比例的读段后，每个样本平均获得51.9 Mb干净读段。随后采用HISAT2软件将干净读段比对至参考基因组，平均比对率达92.91%，各样本比对结果的一致性表明样本间具有可比性。RNA-seq数据比较采用每百万片段每千碱基片段数（FPKM）进行标准化。
实验结果：在携带APPswe突变的成骨细胞前体中，共鉴定出917个上调基因与1825个下调基因。其中，154个上调基因及269个下调基因编码分泌型蛋白。值得注意的是，基因本体（GO）富集分析显示，大部分上调基因参与炎症反应、细胞因子产生及细胞因子/趋化因子介导的信号通路；而大部分下调基因则与细胞周期、细胞增殖及骨矿化过程相关。
研究结论：携带APPswe突变的成骨细胞前体中细胞因子与趋化因子水平升高，提示此类细胞可能发生细胞衰老。
实验整体设计：6月龄OCN-Cre;Ai9及TgAPPsweOCN;OCN-Cre;Ai9小鼠的培养骨髓间充质干细胞mRNA表达谱。