Deciphering the dynamic characteristics of non-neuronal cells in dorsal root ganglion of rat at different developmental stage based on single cell transcriptome data / 生物工程学报

Dorsal root ganglia (DRG) is an essential part of the peripheral nervous system and the hub of the peripheral sensory afferent. The dynamic changes of neuronal cells and their gene expression during the development of dorsal root ganglion have been studied through single-cell RNAseq analysis, while the dynamic changes of non-neuronal cells have not been systematically studied. Using single cell RNA sequencing technology, we conducted a research on the non-neuronal cells in the dorsal root ganglia of rats at different developmental stage. In this study, primary cell suspension was obtained from using the dorsal root ganglions (DRGs, L4-L5) of ten 7-day-old rats and three 3-month-old rats. The 10×Genomics platform was used for single cell dissociation and RNA sequencing. Twenty cell subsets were acquired through cluster dimension reduction analysis, and the marker genes of different types of cells in DRG were identified according to previous researches about DRG single cell transcriptome sequencing. In order to find out the non-neuronal cell subsets with significant differences at different development stage, the cells were classified into different cell types according to markers collected from previous researches. We performed pseudotime analysis of 4 types Schwann cells. It was found that subtype Ⅱ Schwann cells emerged firstly, and then were subtype Ⅲ Schwann cells and subtype Ⅳ Schwann cells, while subtype Ⅰ Schwann cells existed during the whole development procedure. Pseudotime analysis indicated the essential genes influencing cell fate of different subtypes of Schwann cell in DRG, such as Ntrk2 and Pmp2, which affected cell fate of Schwann cells during the development period. GO analysis of differential expressed genes showed that the up-regulated genes, such as Cst3 and Spp1, were closely related to biological process of tissue homeostasis and multi-multicellular organism process. The down regulated key genes, such as Col3a1 and Col4a1, had close relationship with the progress of extracellular structure organization and negative regulation of cell adhesion. This suggested that the expression of genes enhancing cell homestasis increased, while the expression of related genes regulating ECM-receptor interaction pathway decreased during the development. The discovery provided valuable information and brand-new perspectives for the study on the physical and developmental mechanism of Schwann cell as well as the non-neuronal cell changes in DRG at different developmental stage. The differential gene expression results provided crucial references for the mechanism of somatosensory maturation during development.

Transcriptomic profiles of paraquat-induced Parkinson-like changes in mouse brains based on single-cell RNA sequencing / 环境与职业医学

Background Paraquat (PQ) is one of the most widely used herbicides in the world and a risk factor for Parkinson's disease (PD), but the mechanisms underlying PD are poorly understood. Single-cell RNA sequencing (scRNA-seq) technology can study cellular heterogeneity at genetic level, providing insights into the pathogenesis of PQ-induced PD. Objective To analyze the brain cell grouping of PQ-infected mice and the biological processes involved in the subpopulation of PD-like changes cells by scRNA-seq, and to provide clues for revealing potential mechanisms of PQ-induced PD-like changes in mouse brains. Methods Six male 6-week-old C57BL/6 mice were randomly divided into a control group and an experimental group, three mice in each group, and were intraperitoneally injected with 0 (saline) and 10.0 mg·kg−1 PD respectively, once every two days, for 10 consecutive injections for modeling. After infection, mouse brains were taken and scRNA-seq was performed. Cell segmentation was performed according to gene expression characteristics of different cell types, PD-related cell subsets were screened by bioinformatics tools, and gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), protein interaction network analysis, and transcription factor prediction were performed on their characteristic genes. Finally, GO and KEGG analyses were performed on the differential genes of PD-associated cell subsets between the PQ-treated group and the control group, and the biological processes in which these genes may participate were analyzed. Results The sequencing data met quality control standards, a total of 55779 cells were obtained, and all cell dimensionality reduction analysis results showed that they could be further divided into 37 clusters, including 5 major cell types. Based on the KEGG analysis of the top 20 characteristic genes of each subpopulation, the specifically expressed Cluster 33 subpopulation (dopaminergic neurons) was screened and found to be significantly associated with PD. The results of GO analysis showed that the biological function of this subpopulation mainly enriched neurotransmitter transport and regulation. The results of GSEA analysis showed that the tyrosine metabolic pathway and the ligand-receptor interaction pathway of neural activity in brain tissues were significantly enriched. The analysis of transcriptional regulatory networks showed that 39 transcription factors were expressed differently. The metabolic pathway of the dopamine neuronal subset, endocytosis, Ras-associated protein 1 (Rap1) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway were all affected by PQ exposure, according to further analysis of its effects on this subpopulation. The GO analysis showed that differential genes were involved in biological processes such as ion transport and synaptic assembly regulation, and were involved in the cellular component formation of cytoplasm and synapses. Conclusion This study has initially mapped the transcriptome of single cells in the mouse brain after PQ exposure, and screened out the specific expression of Cluster 33 subgroup (dopaminergic neurons), which is significantly correlated with PD, and its biological function changes may be one of the mechanisms of PD-like changes in the mouse brain induced by PQ.

Specific expression of FABP5 in type II alveolar epithelial cells in silicosis pulmonary fibrosis based on single-cell transcriptome sequencing / 环境与职业医学

Background Silicosis is caused by long-term inhalation of large amounts of free silica (SiO2) particles, and exploring its mechanism can provide new directions for the treatment of silicosis fibrosis. Objective To investigate the expression and role of fatty acid binding protein 5 (FABP5) in a silica-induced silicosis model. Methods In combination with the results of single-cell transcriptome sequencing, the expression pattern of FABP5 in mouse alveolar epithelial cells was explored by bioinformatic analysis, and the distribution pattern of fabp5 was detected by spatial transcriptomics. An in vivo model of silicosis was established by intratracheal injection with SiO2 into mice and four groups were set up: normal saline (NS) 7 d group, NS 56 d group, SiO2 7 d group, and SiO2 56 d group. An in vitro model of silicosis was established in SiO2-treated mouse lung epithelial cell line (MLE-12). At the whole animal level, the marker of epithelial cells (E-Cad) and the protein level of FABP5 were detected by tissue immunofluorescence assay; in vitro, the changes of fabp5 mRNA expression and protein level in MLE-12. Results The results of single-cell transcriptome sequencing and spatial transcriptome sequencing showed that the mRNA expression of fabp5 was upregulated in type II alveolar epithelial cells in the focal area of silicosis in mice, accompanied by elevated tissue immunofluorescent protein levels, and there was co-localization of E-CAD. Meanwhile, SiO2 stimulation induced a 1.58-fold increase in fabp5 mRNA expression and a 2-fold increase in protein levels in MLE-12 cells, with significant differences (P<0.05). Conclusion The protein level of FABP5 is increased in alveolar epithelial cells in a pulmonary fibrosis model, suggesting that FABP5 may be involved in the pathological process of epithelial cells in pulmonary fibrosis.

A Single-cell Transcriptome Atlas of Cashmere Goat Hair Follicle Morphogenesis / 基因组蛋白质组与生物信息学报·英文版

Cashmere, also known as soft gold, is produced from the secondary hair follicles (SHFs) of cashmere goats. The number of SHFs determines the yield and quality of cashmere; therefore, it is of interest to investigate the transcriptional profiles present during cashmere goat hair follicle development. However, mechanisms underlying this development process remain largely unexplored, and studies regarding hair follicle development mostly use a murine research model. In this study, to provide a comprehensive understanding of cellular heterogeneity and cell fate decisions, single-cell RNA sequencing was performed on 19,705 single cells of the dorsal skin from cashmere goat fetuses at induction (embryonic day 60; E60), organogenesis (E90), and cytodifferentiation (E120) stages. For the first time, unsupervised clustering analysis identified 16 cell clusters, and their corresponding cell types were also characterized. Based on lineage inference, a detailed molecular landscape was revealed along the dermal and epidermal cell lineage developmental pathways. Notably, our current data also confirmed the heterogeneity of dermal papillae from different hair follicle types, which was further validated by immunofluorescence analysis. The current study identifies different biomarkers during cashmere goat hair follicle development and has implications for cashmere goat breeding in the future.

Application prospects of single-cell transcriptome sequencing in traditional Chinese medicine research / 中国中药杂志

Single-cell transcriptome sequencing(scRNA-seq) can be used to analyze the expression characteristics of the transcriptome at the level of individual cell, and discover the heterogeneity of gene expression in individual cell that is "diluted" or averaged in study of group organization. The scRNA-seq, with the characteristics of standardization, high-throughput, and high integration, can greatly simplify the experimental operation and significantly reduce the consumption of reagents. At the same time, a variety of cells are screened and the gene expression patterns are analyzed at the single-cell level to provide a more efficient detection technique and more rich and accurate information for drug research. In the field of traditional Chinese medicine(TCM), the scRNA-seq is still a new technology, but the individual and precision concepts embodied by scRNA-seq and the theory of TCM syndrome differentiation and treatment have reached the same effect between the micro and macro aspects. This study tried to broaden the thinking for the modernization of TCM by introducing the development of scRNA-seq technology and its application in modern drug research and discussing the application prospects of scRNA-seq in TCM research.

Methods for mammalian single cell research - a review / 生物工程学报

Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.

Single-cell transcriptomics reveals gene signatures and alterations associated with aging in distinct neural stem/progenitor cell subpopulations

Aging associated cognitive decline has been linked to dampened neural stem/progenitor cells (NSC/NPCs) activities manifested by decreased proliferation, reduced propensity to produce neurons, and increased differentiation into astrocytes. While gene transcription changes objectively reveal molecular alterations of cells undergoing various biological processes, the search for molecular mechanisms underlying aging of NSC/NPCs has been confronted by the enormous heterogeneity in cellular compositions of the brain and the complex cellular microenvironment where NSC/NPCs reside. Moreover, brain NSC/NPCs themselves are not a homogenous population, making it even more difficult to uncover NSC/NPC sub-type specific aging mechanisms. Here, using both population-based and single cell transcriptome analyses of young and aged mouse forebrain ependymal and subependymal regions and comprehensive "big-data" processing, we report that NSC/NPCs reside in a rather inflammatory environment in aged brain, which likely contributes to the differentiation bias towards astrocytes versus neurons. Moreover, single cell transcriptome analyses revealed that different aged NSC/NPC subpopulations, while all have reduced cell proliferation, use different gene transcription programs to regulate age-dependent decline in cell cycle. Interestingly, changes in cell proliferation capacity are not influenced by inflammatory cytokines, but likely result from cell intrinsic mechanisms. The Erk/Mapk pathway appears to be critically involved in regulating age-dependent changes in the capacity for NSC/NPCs to undergo clonal expansion. Together this study is the first example of using population and single cell based transcriptome analyses to unveil the molecular interplay between different NSC/NPCs and their microenvironment in the context of the aging brain.