Single cell RNA-sequencing of lineage-negative fraction of stromal cells isolated from inguinal white adipose tissue in mouse

A subset of adipocytes residing within the inguinal white adipose tissue (ingWAT) of mice exhibit thermogenic activity in response to various external stimuli, including cold exposure. The inducible nature of this thermogenic response, coupled with its robust energy-depleting capacity have prompted investigation into the adipose precursor cells (APCs) from which thermogenic adipocytes derive. To this end, we performed single-cell transcriptomics on cells derived from ingWAT, interscapular brown adipose tissue (iBAT) and epididymal WAT. A subset of single cells collected from ingWAT and epiWAT of mice were chronically (4 days) treated with CL-316,243 (dose at 1 mg kg -1). Tissues of n=28, 10 week-old mice were digested and stromal cells were subsequently purified via differential centrifugation. Single-cell RNA extraction and mRNA amplification were performed on the C1™ Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) following the protocol (PN 100-7168, http://www.fluidigm.com/). Following centrifugation and removal of the medium, cells were resuspended at a concentration of 150–500 cells/µL. This cell suspension was mixed with C1 Cell Suspension Reagent (Fluidigm, Cat # 634833) at the recommended ratio of 3:2 immediately before loading 5 µL of this final mix on the C1 IFC. We obtained, on average, 2.5 million mapped reads per one single cell and successfully reconstructed single-cell expression of ~9,000 genes. Our results revealed a unique cluster of cells that exhibited enriched expression of canonical thermogenic and adipogenic gene markers. Notably, we identified tetraspanin CD81 as a discretely expressed membrane-bound protein conserved specifically within this population. All experiments were performed at our facility at the University of California, San Francisco.

小鼠腹股沟白色脂肪组织（inguinal white adipose tissue, ingWAT）内存在一类脂肪细胞亚群，可响应包括寒冷暴露在内的多种外界刺激，展现出产热活性。该产热反应具有可诱导性，且具备强大的能量消耗能力，这促使学界对产热脂肪细胞的来源——脂肪前体细胞（adipose precursor cells, APCs）展开研究。为此，我们对源自小鼠腹股沟白色脂肪组织、肩胛间棕色脂肪组织（interscapular brown adipose tissue, iBAT）以及附睾白色脂肪组织的细胞开展了单细胞转录组学分析。我们对取自小鼠腹股沟白色脂肪组织与附睾白色脂肪组织的部分单细胞，采用CL-316,243（剂量为1 mg·kg⁻¹）进行了4天的慢性处理。我们对28只10周龄小鼠的组织进行消化，随后通过差速离心纯化基质细胞。依照产品手册（PN 100-7168，http://www.fluidigm.com/）的操作流程，使用C1™单细胞自动制备集成流体回路（Integrated Fluidic Circuit, IFC）完成单细胞RNA提取与mRNA扩增。经离心并弃去培养基后，将细胞重悬至150~500个细胞/μL的浓度。在上样前，即刻按照推荐的3:2比例，将该细胞悬浮液与C1细胞悬浮液试剂（Fluidigm，货号634833）混合，随后取5 μL该最终混合液上样至C1 IFC。我们平均每个单细胞获得250万条比对读数，并成功重建了约9000个基因的单细胞表达谱。分析结果显示，存在一类独特的细胞簇，其显著富集经典产热与成脂基因标志物。值得注意的是，我们鉴定出四跨膜蛋白CD81是该细胞群特异性表达的膜结合蛋白，且在该群体中保守存在。本研究所有实验均于加州大学旧金山分校的实验平台完成。