Single-cell RNA sequencing of myeloid cells, hematopoietic stem and progenitor cells, and mesenchymal stromal cells isolated from bone marrow of C57BL/6J WT and IL-1rn-KO mice

Interleukin-1ß (IL-1ß) drives hematopoietic stem cell (HSC) differentiation into the myeloid lineage, and enhanced IL-1ß signaling plays a key role in hematological malignancies. However, little is known on the role of its endogenous regulatory cytokine, IL-1 receptor antagonist (IL-1rn), on both healthy and malignant hematopoiesis. Here, characterize transcriptomic changes at the single cell level in myeloid cells, hematopoietic stem and progenitor cells, and CD63+ mesenchymal stromal cells between C57BL/6J and IL-1rn-KO mice. Overall design: We profiled bone marrow (BM) hematopoietic and non-hematopoietic cells by scRNA-seq from females C57BL6/J WT (n = 1) and IL-1RN KO (n = 1) aged 13-15 weeks. We sorted myeloid cells as CD11b+, hematopoietic stem and progenitor cells (HSPC) as Lineage- Sca-1+ cKit+ (LSK) and mesenchymal stromal cells (MSC) as CD45- CD31- Ter119- CD63+ by FACS. Sorted CD63+ MSCs were pooled from two animals to reach the desired number of cells for scRNA-seq, after separation of one femur and one tibia from one of the mice dedicated to CD11b+ and LSK cell sorting. Cells were counted and their viability checked using the Countess III cell counter (Thermofisher). Single cells were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics). Each cell suspension was loaded into one port of a Chromium Next GEM Chip G (10x Genomics) with a target output of 4000-10000 cells.

白细胞介素1β（Interleukin-1β, IL-1β）可驱动造血干细胞（hematopoietic stem cell, HSC）向髓系分化，而增强的IL-1β信号通路在血液系统恶性肿瘤中发挥关键作用。然而，其内源调控性细胞因子——白细胞介素1受体拮抗剂（IL-1 receptor antagonist, IL-1rn）在健康与恶性造血过程中的功能尚不清楚。本研究对C57BL/6J小鼠与IL-1rn基因敲除（IL-1rn-KO）小鼠的髓系细胞、造血干祖细胞及CD63阳性间充质基质细胞进行单细胞水平转录组变化分析。
整体实验设计：我们对13-15周龄的雌性C57BL/6J野生型（WT，n = 1）与IL-1RN敲除（KO，n = 1）小鼠的骨髓（bone marrow, BM）造血与非造血细胞开展单细胞RNA测序（scRNA-seq）。通过荧光激活细胞分选术（FACS）分选出CD11b阳性髓系细胞、谱系阴性-Sca-1阳性-cKit阳性（Lineage⁻ Sca-1⁺ cKit⁺, LSK）造血干祖细胞（hematopoietic stem and progenitor cells, HSPC），以及CD45⁻CD31⁻Ter119⁻CD63⁺间充质基质细胞（mesenchymal stromal cells, MSC）。其中，CD63阳性MSCs需从两只动物中混合以达到单细胞RNA测序所需的细胞量：我们从一只用于分选CD11b⁺与LSK细胞的小鼠中分离单侧股骨与胫骨后获取细胞并混合。随后使用Countess III细胞计数器（赛默飞世尔, Thermofisher）对细胞进行计数并检测活力。采用Chromium控制器（10x Genomics公司）将单细胞包裹至乳液微滴中。将每份细胞悬液加载至Chromium Next GEM G型芯片（10x Genomics公司）的一个孔道中，目标捕获细胞数为4000-10000个。