10X sequencing of immune cells and limb regenerates

10X sequencing of immune cells and limb regeneratesImmune 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 = 7,889) and 2) peripheral leukocytes with presumptive macrophages from the intraperitoneal cavity (N = 4,998). 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 = 8,272 and 9,906 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.

免疫细胞与肢体再生组织的10X测序数据集
已知免疫细胞对于墨西哥钝口螈（axolotl, Ambystoma mexicanum）成功实现肢体再生至关重要，但其身份特征、行为模式与功能机制仍有诸多细节有待阐明。本研究从成年墨西哥钝口螈血液中分离外周白细胞，并制备了两份用于单细胞测序的样本：1）外周白细胞（样本量N=7889）；2）取自腹腔的、包含疑似巨噬细胞的外周白细胞（样本量N=4998）。通过k-means聚类分析，我们从每份样本中分别鉴定出6类细胞群，其基因表达特征分别对应红细胞、血栓细胞（thrombocyte）、中性粒细胞、B细胞、T细胞与髓系细胞。第七类疑似巨噬细胞群仅在样本2中被特异性鉴定到。
随后，我们分别在墨西哥钝口螈肢体截肢后1天和6天（截肢后天数，days post-amputation, DPA）分离截肢肢体的细胞，并进行单细胞测序（样本量分别为N=8272和9906个细胞），以鉴定免疫细胞与非免疫细胞群。通过k-means聚类分析，我们共鉴定出8类细胞群，其中多数细胞表达红细胞特异性基因。尽管红细胞占比极高，我们仍通过无偏分析方法在两个时间点均鉴定出浸润性中性粒细胞、巨噬细胞与淋巴细胞群。此外，我们还鉴定出表达表皮细胞、成纤维样细胞与内皮细胞特征基因的细胞群。
与既往实验研究结果一致，中性粒细胞在截肢后1天的丰度高于6天，而巨噬细胞与非免疫细胞的丰度模式则与之相反。值得注意的是，我们在截肢后1天鉴定到一小群成纤维样细胞，而该细胞群在截肢后6天的细胞占比显著升高。我们推测这类细胞为可分化形成芽基（blastema）的早期祖细胞。本研究得到的富集基因集将为未来探索墨西哥钝口螈肢体再生过程中免疫细胞的多样性与功能的单细胞研究提供助力。