Heterogeneity analysis of astrocytes following spinal cord injury at single-cell resolution

Astrocytes play many important functions in response to spinal cord injury (SCI) in an activated manner, including clearance of necrotic tissue, formation of protective barrier, maintenance of microenvironment balance, interaction with immune cells, and formation of the glial scar. More and more studies have shown that the astrocytes are heterogeneous, such as inflammatory astrocyte 1 (A1) and neuroprotective astrocyte 2 (A2) types. However, the subtypes of astrocyte resulting from SCI have not been clearly defined. In this study, using single-cell RNA sequencing, we constructed the transcriptomic profile of astrocytes from uninjured spinal cord tissue and nearby the lesion epicenter at 0.5, 1, 3, 7, 14, 60, and 90 days after mouse hemisection spinal cord tissue. Our analysis uncovered six transcriptionally distinct astrocyte states, including Atp1b2+, S100a4+, Gpr84+, C3+/G0s2+, GFAP+/Tm4sf1+, and Gss+/Cryab+ astrocytes. We used these new signatures combined with canonical astrocyte markers to determine the distribution of morphological and physiologically distinct for each astrocyte population at injured sites by immunofluorescence staining. Then we identified the dynamic evolution process of each astrocyte subtype following SCI. Finally, we also revealed the evolution of highly expressed genes in these astrocyte subtypes at different phases of SCI. Together, we provided six astrocyte subtypes at single-cell resolution following SCI. These data not only contribute to understanding the heterogeneity of astrocytes during SCI but also help to find new astrocyte subtypes as a target for SCI repair. Overall design: Single cell RNA sequencing of uninjured and injured spinal cord at pSCI0.5d, pSCI1d, pSCI3d, pSCI7d, pSCI14d, pSCI60d and pSCI90d.

星形胶质细胞在脊髓损伤（spinal cord injury, SCI）后以激活状态发挥诸多重要生物学功能，包括清除坏死组织、形成保护性屏障、维持微环境稳态、与免疫细胞相互作用以及形成胶质瘢痕。越来越多研究表明，星形胶质细胞具有异质性，例如炎性星形胶质细胞1型（A1）与神经保护性星形胶质细胞2型（A2）。但目前由脊髓损伤诱导产生的星形胶质细胞亚型尚未被明确界定。
本研究借助单细胞RNA测序技术，对小鼠半切脊髓损伤模型中未损伤脊髓组织及损伤中心周边组织在损伤后0.5、1、3、7、14、60及90天的星形胶质细胞转录组图谱进行构建。分析结果显示存在六种转录层面特征显著不同的星形胶质细胞状态，分别为Atp1b2+、S100a4+、Gpr84+、C3+/G0s2+、GFAP+/Tm4sf1+以及Gss+/Cryab+星形胶质细胞。本研究利用上述新特征基因结合经典星形胶质细胞标志物，通过免疫荧光染色技术明确了损伤位点各星形胶质细胞群体在形态与生理功能上的差异分布；随后解析了脊髓损伤后各星形胶质细胞亚型的动态演化进程，并揭示了不同损伤阶段这些星形胶质细胞亚型的高表达基因演化规律。
本研究最终明确了脊髓损伤后单细胞分辨率下的六种星形胶质细胞亚型。该数据集不仅有助于理解脊髓损伤过程中星形胶质细胞的异质性，还为寻找脊髓损伤修复的新型星形胶质细胞靶点提供了重要支撑。
整体实验设计：对未损伤脊髓组织及脊髓损伤后0.5天（pSCI0.5d）、1天（pSCI1d）、3天（pSCI3d）、7天（pSCI7d）、14天（pSCI14d）、60天（pSCI60d）及90天（pSCI90d）的损伤脊髓组织进行单细胞RNA测序。