Isolation and adipogenic differentiation of murine mesenchymal stem cells harvested from macrophage-depleted bone marrow and adipose tissue

Mouse mesenchymal stem cells (MSCs) provide a resourceful tool to study physiological and pathological aspects of adipogenesis. Bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (ASCs) are widely used for these studies. Since there is a wide spectrum of methods available, the purpose is to provide a focused hands-on procedural guide for isolation and characterization of murine BM-MSCs and ASCs and to effectively differentiate them into adipocytes. Optimized harvesting procedures for murine BM-MSCs and ASCs are described and graphically documented. Since macrophages reside in bone-marrow and fat tissues and regulate the biological behaviour of BM-MSCs and ASCs, we included a procedure to deplete macrophages from the MSC preparations. The identity and stemness of BM-MSCs and ASCs were confirmed by flow cytometry using established markers. Since the composition and concentrations of adipogenic differentiation cocktails differ widely, we present a standardized four-component adipogenic cocktail, consisting of insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and indomethacin to efficiently differentiate freshly isolated or frozen/thawed BM-MSCs and ASCs into adipocytes. We further included visualization and quantification protocols of the differentiated adipocytes. This laboratory protocol was designed as a step-by-step procedure for harvesting murine BM-MSCs and ASCs and differentiating them into adipocytes.

小鼠间充质干细胞（Mouse mesenchymal stem cells, MSCs）是研究脂肪生成生理与病理过程的宝贵实验工具。骨髓源性间充质干细胞（bone marrow-derived MSCs, BM-MSCs）与脂肪组织源性间充质干细胞（adipose tissue-derived MSCs, ASCs）是此类研究中应用最为广泛的细胞模型。鉴于当前可选用的实验方法种类繁多，本指南旨在提供一套聚焦的实操流程方案，用于小鼠BM-MSCs与ASCs的分离、鉴定，并高效诱导其向脂肪细胞分化。
本指南详细描述了优化后的小鼠BM-MSCs与ASCs取材流程，并配有图文记录。由于巨噬细胞定居于骨髓与脂肪组织中，可调控BM-MSCs与ASCs的生物学行为，因此本指南纳入了从间充质干细胞制剂中清除巨噬细胞的操作步骤。研究人员通过流式细胞术使用已验证的细胞标志物，确认了BM-MSCs与ASCs的细胞身份及干细胞干性。
鉴于脂肪分化诱导液的组分与浓度差异极大，本指南提供了一套标准化的四组分脂肪分化诱导液配方，其组分为胰岛素、地塞米松、3-异丁基-1-甲基黄嘌呤与吲哚美辛，可高效将新鲜分离或冻存复苏后的BM-MSCs与ASCs诱导分化为脂肪细胞。此外，本指南还补充了分化后脂肪细胞的可视化与定量分析方案。本实验室方案采用分步式流程设计，用于小鼠BM-MSCs与ASCs的取材及向脂肪细胞的分化诱导。