Single Cell Sequencing Reveals Gene Expression Signatures Associated with Bone Marrow Stromal Cell Subpopulations and Time in Culture [NGS_bulk cell RNA-seq]. Single Cell Sequencing Reveals Gene Expression Signatures Associated with Bone Marrow Stromal Cell Subpopulations and Time in Culture [NGS_bulk cell RNA-seq]

Background Bone marrow stromal cells (BMSCs) are a heterogeneous population that participates in wound healing, immune modulation and tissue regeneration. Next generation sequencing was used to analyze transcripts from single BMSCs in order to better characterize BMSC subpopulations. Methods Cryopreserved passage 2 BMSCs from one healthy subject were cultured through passage 10. The transcriptomes of bulk BMSCs from designated passages were analyzed with microarrays and RNA sequencing (RNA-Seq). For some passages, single BMSCs were separated using microfluidics and their transcriptomes were analyzed by RNA-Seq. Results Transcriptome analysis microarray and RNA-Seq of unseparated BMSCs from passages 2, 4, 6, 8, 9 and 10 yielded similar results; both data sets grouped passages 4 and 6 and passages 9 and 10 together and genes differentially expressed among these early and late passage BMSCs were similar. 3D Diffusion map visualization of single BMSCs from passages 3, 4, 6, 8 and 9 clustered passage 3 and 9 into two distinct groups, but there was considerable overlap for passage 4, 6 and 8 cells. Markers for early passage, FGFR2, and late passage BMSCs, PLAT, were able to identify three subpopulations within passage 3 BMSCs; one that expressed high levels of FGFR2 and low levels of PLAT; one that expressed low levels of FGFR2 and high levels of PLAT and one that expressed intermediate levels of FGFR2 and low levels of PLAT. Conclusions Single BMSCs can be separated by microfluidics and their transcriptome analyzed by next generation sequencing. Single cell analysis of early passage BMSCs identified a subpopulation of cells expressing high levels of FGFR2 that might include skeletal stem cells. Overall design: Cryopreserved passage 2 BMSCs from one healthy subject were cultured through passage 10. The transcriptomes of bulk BMSCs from designated passages were analyzed with microarrays and RNA sequencing (RNA-Seq). For some passages, single BMSCs were separated using microfluidics and their transcriptomes were analyzed by RNA-Seq.

背景 骨髓基质细胞（Bone marrow stromal cells, BMSCs）是一类异质性细胞群，参与伤口愈合、免疫调节与组织再生过程。为更精准地表征BMSCs的亚群特征，本研究采用下一代测序技术（Next generation sequencing）对单个BMSCs的转录本进行分析。

方法 从1名健康供体获取的冻存第2代BMSCs被培养至第10代。对指定传代次数的批量BMSCs转录组，分别采用微阵列与RNA测序（RNA sequencing, RNA-Seq）进行分析。针对部分传代的细胞，采用微流控技术分离单个BMSCs，并通过RNA-Seq分析其转录组。

结果 对第2、4、6、8、9、10代未分离的BMSCs进行转录组微阵列和RNA-Seq分析，得到了一致性结果；两组数据集均将第4代与第6代、第9代与第10代细胞聚为一类，且早代与晚代BMSCs间的差异表达基因谱相似。对第3、4、6、8、9代单个BMSCs的三维扩散图可视化分析显示，第3代与第9代细胞各自聚为独立的细胞簇，但第4、6、8代细胞存在显著的重叠。早代BMSCs标志物FGFR2与晚代BMSCs标志物PLAT可区分第3代BMSCs中的3个亚群：分别为高表达FGFR2且低表达PLAT的亚群、低表达FGFR2且高表达PLAT的亚群，以及FGFR2表达水平中等且低表达PLAT的亚群。

结论 可通过微流控技术分离单个BMSCs，并采用下一代测序技术分析其转录组。对早代BMSCs的单细胞分析发现了一个高表达FGFR2的细胞亚群，该亚群可能包含骨骼干细胞。

整体实验设计：从1名健康供体获取的冻存第2代BMSCs被培养至第10代。对指定传代次数的批量BMSCs转录组，分别采用微阵列与RNA测序（RNA-Seq）进行分析。针对部分传代的细胞，采用微流控技术分离单个BMSCs，并通过RNA-Seq分析其转录组。