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（由申请人提供）：先前对脊髓损伤（SCI）修复的努力集中于通过解除髓磷脂组分的抑制来促进切断的神经的长距离生长或增强CNS神经元的先天再生能力。最近，越来越多的证据指出了一种替代方法，即，通过引入/激活外源性/内源性神经干细胞（NSC），允许产生新的神经元，其形成新生神经回路，作为连接来自上行和下行神经的信号的中继站，以实现功能恢复。一个主要障碍是CNS损伤环境，例如脊髓损伤（SCI）后的环境，是相当抑制性和炎症性的，限制了活化的NSC分化成神经元的能力。同时，NSC的稀缺性及其周围环境的复杂性使得这些细胞的分子表征特别具有挑战性，因为具有异质细胞组成的组织的RNAseq分析提供了此类组织中所有不同细胞的总和或平均值，这通常提供与NSC活性无关的无意误导性读数。利用本实验室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区CD 133（由CD 133基因编码）阳性室管膜细胞的静止NSC特征。使用ROSA 26-Td-番茄报告小鼠，电穿孔了Cre启动子驱动的Cre，我们进一步证明，在VEGF和bFGF刺激下，可能与损伤的情况一样，CD 133+室管膜细胞可以被有丝分裂激活并分化为下游神经谱系细胞（MAP 2+神经元和GFAP+神经胶质细胞），甚至在第四脑室的室管膜/心室表面，这在出生后的小鼠体内还没有被报道为神经源性的。在本申请中，我们建议通过将B6 N; 129 S-Prom 1 tm 1（cre/ERT 2）Gilb/J（杰克逊实验室）小鼠与Cre-LoxP报告小鼠Rosa 26-Td-番茄杂交来追踪CD 133后代，以研究T9-11挤压损伤后脊髓中央管内衬的CD 133+室管膜细胞的增殖、迁移和分化。基于时间和空间活动 为了研究SCI后CD 133阳性室管膜细胞的分子生物学特性，我们在SCI前后的不同时间点和不同区域对CD 133阳性室管膜细胞进行单细胞转录组分析，并采用加权基因共表达网络分析（WGCNA）对SCI后CD 133阳性室管膜细胞的分子生物学特性和活性进行分析。我们相信，这项研究将有助于未来发展新的治疗策略，以加强内源性神经干细胞介导的再生后SCI。

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