Using single cell RNAseq to study stem cell activity after spinal cord injury

使用单细胞 RNAseq 研究脊髓损伤后干细胞活性

• 批准号: 9148090
• 负责人: YI EVE SUN
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9148090
• 关键词: Adult; Attenuated; Automobile Driving; Behavior; Biological; Biological Assay; Biological Neural Networks; Cell Line; Cell Lineage; Cell Therapy; Cells; Central cord canal structure; Complex; Coupled; Cre-LoxP; Crush Injury; DNA cassette; Data; Data Analyses; Development; Differentiation Antigens; Electroporation; Enhancers; Environment; Ependymal Cell; FGF2 gene; Future; Generations; Genes; Glial Fibrillary Acidic Protein; Growth; In Situ Hybridization; Inflammatory; Informatics; Injury; Label; Lateral; Life; Light; Mediating; Medical; Mitotic; Molecular; Mus; Myelin; Natural regeneration; Nerve; Neuraxis; Neuroglia; Neuronal Differentiation; Neurons; Pathway Analysis; Peripheral Nervous System; Proliferation Marker; Property; Prosencephalon; Reporter; Reporting; Sampling; Sensory; Signal Pathway; Signal Transduction; Site; Slice; Sorting - Cell Movement; Spinal Cord; Spinal cord injury; Stem cells; Sum; Surface; Tamoxifen; Techniques; Testing; The Jackson Laboratory; Time; Tissues; Tomatoes; Vascular Endothelial Growth Factors; Ventricular; Weight; base; central nervous system injury; differential expression; follow-up; functional restoration; in vivo; injury and repair; migration; motor deficit; nerve stem cell; neural circuit; novel strategies; novel therapeutic intervention; postnatal; programs; promoter; public health relevance; recombinase; regenerative; relating to nervous system; repaired; research study; response; stem; success; transcriptome; transcriptome sequencing

 DESCRIPTION (provided by applicant): Prior efforts towards spinal cord injury (SCI) repair had been focused on promoting long-distance growth of severed nerves by relieving the inhibition from myelin components or to enhance the innate regeneration ability of CNS neurons. Recently, accumulating evidences have pointed out an alternative approach, i.e., through introducing/activation of exogenous/endogenous neural stem cells (NSCs), allowing for generation of new neurons, which form nascent neural circuits, serving as relay stations connecting signals from ascending and descending nerves to achieve functional restoration. One major obstacle is that the CNS injury environment, such as the one after spinal cord injury (SCI), is rather inhibitory and inflammatory, limiting the ability for activated NSCs to differentite into neurons. Meanwhile, the scarcity of NSC and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging, because RNAseq analysis of tissues with heterogeneous cellular composition provide the sum or averaging of all of the different cells in such tissue, which often provide unintentional misleading readouts unrelated to NSC activities. With single cell transcriptome profiling technique established in the lab, we uncovered quiescent NSC features of CD133 (encoded by prominin1 gene) positive ependymal cells in the adult mouse forebrain neurogenic zone. Using ROSA26-Td-tomato reporter mice with electroporation of prominin1 promoter driven Cre, we further demonstrated that upon VEGF and bFGF stimulation, likely as in the case of injury, CD133+ ependymal cells can be mitotically activated and differentiate into downstream neural lineage cells (MAP2+ neurons and GFAP+ glia), even at the ependymal/ventricular surface of the 4th ventricle, which has not been reported to be neurogenic in vivo in postnatal mice. In this application, we propose to trace CD133 progenies by crossing the B6N;129S-Prom1tm1(cre/ERT2)Gilb/J (the Jackson Lab) mice with the Cre-LoxP reporter mice, Rosa26-Td-tomato to study proliferation, migration and differentiation of CD133+ ependymal cells lining the spinal cord central canal after crush injury at T9-11. Based on temporal and spatial activities of CD133 progenies after SCI, we will perform single cell transcriptome profiling on CD133 + ependymal cell at several regions and time points before and after SCI, followed by weighted gene co-expression network analysis (WGCNA) to characterize the molecular features and activaties of CD133+ ependymal cells after SCI. We believe such study will be instrumental for future development of the new therapeutic strategies to enhance endogenous NSC mediated regeneration after SCI.

 描述（通过应用提供）：通过缓解髓磷脂成分的抑制或增强CNS神经元的先天再生能力，提高了脊髓损伤（SCI）修复的事先努力（SCI）修复。最近，积累的证据指出了一种替代方法，即通过引入/激活外源/内源性神经元干细胞（NSC），允许产生新的神经元，该神经元形成新的神经元，形成新的神经元，用作接力站，从而连接到升高和降序神经实现功能恢复的信号。一个主要的障碍是，CNS损伤环境（例如脊髓损伤后的损伤环境（SCI））是抑制性和炎症性的，限制了激活NSC将不同岩具有不同神经元进入神经元的能力。同时，NSC的稀缺性及其周围环境的复杂性使这些细胞的分子表征特别挑战，因为RNASEQ对具有异质性细胞组成的组织分析提供了这种组织中所有不同细胞的总和或平均，这通常提供了与NSC活动无关的无关误导性读数。通过实验室中建立的单细胞转录组分析技术，我们发现了成年小鼠前脑神经源性区中CD133（由Prominin1基因编码）的静态NSC特征。 Using ROSA26-Td-tomato reporter mice with electroporation of prominin1 promoter driven Cre, we further demonstrated that upon VEGF and bFGF stimulation, likely as in the case of injury, CD133+ ependymal cells can be mitotically activated and differentiate into downstream neuronal lineage cells (MAP2+ neurons and GFAP+ glia), even at the ependymal/ventricular surface of第四个心室，据报道我们在产后小鼠的体内是神经源。在此应用中，我们提议通过越过B6N; 129S-PROM1TM1（CRE/ERT2）GILB/J（Jackson Lab）小鼠与Cre-loxP报告基因小鼠，Rosa26-TD-TOMATO研究，研究了CD133+ Ependent in ninding the At the At the At the At the At the At the At the Ependinsal condine spintials cre-loxp小鼠，以追踪CD133的祖子。基于临时和空间活动 在SCI后CD133后代，我们将在SCI之前和之后的几个区域和时间点上对CD133 + dend症细胞进行单细胞转录组分析，然后进行加权基因共表达网络分析（WGCNA），以表征SCI后CD133 + epentymal细胞的分子特征和活性。我们认为，此类研究将有助于未来开发新的治疗策略，以增强SCI后内源性NSC介导的再生。

项目成果

Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation.

耦合电生理记录和单细胞转录组分析揭示了神经元成熟的分子机制

• DOI: 10.1007/s13238-016-0247-8
• 发表时间: 2016-03
• 期刊: Protein & cell
• 影响因子: 21.1
• 作者: Chen X;Zhang K;Zhou L;Gao X;Wang J;Yao Y;He F;Luo Y;Yu Y;Li S;Cheng L;Sun YE
• 通讯作者: Sun YE

Unbiased transcriptomic analyses reveal distinct effects of immune deficiency in CNS function with and without injury.

无偏见的转录组分析揭示了免疫缺陷对有损伤和无损伤的中枢神经系统功能的明显影响

• DOI: 10.1007/s13238-018-0559-y
• 发表时间: 2019-08
• 期刊: Protein & cell
• 影响因子: 21.1
• 作者: Luo D;Ge W;Hu X;Li C;Lee CM;Zhou L;Wu Z;Yu J;Lin S;Yu J;Xu W;Chen L;Zhang C;Jiang K;Zhu X;Li H;Gao X;Geng Y;Jing B;Wang Z;Zheng C;Zhu R;Yan Q;Lin Q;Ye K;Sun YE;Cheng L
• 通讯作者: Cheng L