Identification of immune and non-immune cells in regenerating axolotl limbs by single-cell sequencing
Immune cells are known to be critical for successful limb regeneration in the axolotl (Ambystoma mexicanum), but many details regarding their identity, behavior, and function are yet to be resolved. We isolated peripheral leukocytes from the blood of adult axolotls and then created two samples for single-cell sequencing: 1) peripheral leukocytes (N = 7889) and 2) peripheral leukocytes with presumptive macrophages from the intraperitoneal cavity (N = 4998). Using k-means clustering, we identified 6 cell populations from each sample that presented gene expression patterns indicative of erythrocyte, thrombocyte, neutrophil, B-cell, T-cell, and myeloid cell populations. A seventh, presumptive macrophage cell population was identified uniquely from sample 2. We then isolated cells from amputated axolotl limbs at 1 and 6 days post-amputation (DPA) and performed single cell sequencing (N = 8272 and 9906 cells respectively) to identify immune and non-immune cell populations. Using k-means clustering, we identified 8 cell populations overall, with the majority of cells expressing erythrocyte-specific genes. Even though erythrocytes predominated, we used an unbiased approach to identify infiltrating neutrophil, macrophage, and lymphocyte populations at both time points. Additionally, populations expressing genes for epidermal cells, fibroblast-like cells, and endothelial cells were also identified. Consistent with results from previous experimental studies, neutrophils were more abundant at 1 DPA than 6 DPA, while macrophages and non-immune cells exhibited inverse abundance patterns. Of note, we identified a small population of fibroblast-like cells at 1 DPA that was represented by considerably more cells at 6 DPA. We hypothesize that these are early progenitor cells that give rise to the blastema. The enriched gene sets from our work will aid future single-cell investigations of immune cell diversity and function during axolotl limb regeneration. Keywords: 10X genomics; Axolotl; Gene expression; Limb regeneration; Single-cell.
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基因水平:PCR Array、RT-PCR、PCR、单细胞测序
蛋白水平:MSD、Luminex、CBA、Elispot、Antibody Array、ELISA、Sengenics
细胞水平:细胞染色、细胞分选、细胞培养、细胞功能
组织水平:空间多组学、多重荧光免疫组化、免疫组化、免疫荧光
数据分析:流式数据分析、组化数据分析、多因子数据分析
基因水平:PCR Array、RT-PCR、PCR、单细胞测序
蛋白水平:MSD、Luminex、CBA、Elispot、Antibody Array、ELISA、Sengenics
细胞水平:细胞染色、细胞分选、细胞培养、细胞功能
组织水平:空间多组学、多重荧光免疫组化、免疫组化、免疫荧光
数据分析:流式数据分析、组化数据分析、多因子数据分析
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