Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health (NIH), 10 Center Drive-MSC-1184, Building 10, Room 3C720, Bethesda, MD, 20892-1184, USA.
Biometric Research Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, MD, USA.
J Transl Med. 2019 Jan 11;17(1):23. doi: 10.1186/s12967-018-1766-2.
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.
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.
Transcriptome analysis by 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 passages 3 and 9 into two distinct groups, but there was considerable overlap for passages 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.
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.
骨髓基质细胞（BMSCs）是一种异质群体，参与伤口愈合、免疫调节和组织再生。下一代测序技术被用于分析来自单个 BMSCs 的转录本，以便更好地描述 BMSC 亚群。
从一位健康供体中分离出冷冻保存的第 2 代 BMSCs，并通过第 10 代培养。使用微阵列和 RNA 测序（RNA-Seq）分析指定代次的 BMSCs 群体的转录组。对于某些代次，使用微流控技术分离单个 BMSCs，并通过 RNA-Seq 分析其转录组。
通过微阵列和 RNA-Seq 对第 2、4、6、8、9 和 10 代未分离的 BMSCs 的转录组分析产生了相似的结果；这两个数据集将第 4 代和第 6 代以及第 9 代和第 10 代分组在一起，而这些早期和晚期 BMSCs 中差异表达的基因相似。第 3、4、6、8 和 9 代的单个 BMSCs 的 3D 扩散图可视化将第 3 代和第 9 代聚类为两个不同的组，但第 4 代、第 6 代和第 8 代细胞之间存在相当大的重叠。早期代次的标志物 FGFR2 和晚期代次的标志物 PLAT 能够鉴定第 3 代 BMSCs 中的三个亚群；一个表达高水平的 FGFR2 和低水平的 PLAT；一个表达低水平的 FGFR2 和高水平的 PLAT；一个表达中间水平的 FGFR2 和低水平的 PLAT。
可以通过微流控技术分离单个 BMSCs，并通过下一代测序技术分析其转录组。早期代次的单个 BMSCs 的单细胞分析鉴定出一个表达高水平 FGFR2 的细胞亚群，该亚群可能包括骨骼干细胞。
