Novel ex vivo culture of purified mouse mesenchymal stromal cells reveals tissue-dependent niche heterogeneity in bone marrow and functional interaction with macrophages

Mesenchymal stem/stromal cells (MSCs) are skeletal stem cells capable of regenerating bone, cartilage, and adipose tissues, while also serving as an essential immunosupportive cell for the hematopoietic stem cell pool. Therefore, the isolation and purification of murine MSCs is essential to understanding the differentiation capabilities necessary for skeletal regeneration, hematopoietic support, and immunomodulatory roles of MSCs within the bone marrow microenvironment (BMME). While many protocols on isolation from bone marrow (BM-MSCs) and bone associated cells (BAC-MSCs) have been published, none achive purity from macropahge contamination and many do not address heterogeneity of MSCs between these two compartments. To ensure a pure MSC isolation, we established a new strategy to exclude macrophages and their precursors by targeting F4/80/Ly6C/CD45. By analyzing MSC transcriptional profiles from both compartments with and without our purification strategy, we demonstrated distinct functional characteristics of MSCs by compartment, as well as functional impacts on the MSC transcriptional profile as a result of macrophage interaction. Overall design: Four populations were collected in total from two different compartments: Bone marrow derived (BM) and bone associated cells (BAC). Bone marrow derived mesenchymal cells (MSC) were harvested from bilateral hind limbs and pelvis whole bone marrow and plated on collagen-I coated plates. After the first passage, samples were either used directly (BM-nonS) or sorted to purely isolate CD31-/CD45-/Ly6C-/F4/80-/CD51+/Sca-1+ cells (BM-S) for bulk RNAseq. BAC derived MSCs were isolated from collagenase-I treated bone fragments from bilateral hind limbs. After harvest these cells were either used directly without depletion (BAC-nonD) or depleted by magnetic separation to remove CD45+/Ly6C+/F4/80+ cells (BAC-D) for bulk RNAseq.

间充质干/基质细胞（Mesenchymal stem/stromal cells, MSCs）是一类兼具骨、软骨及脂肪组织再生能力的骨骼干细胞，同时也是维持造血干细胞池功能不可或缺的免疫支持细胞。因此，分离纯化小鼠MSCs，是解析其在骨髓微环境（bone marrow microenvironment, BMME）中发挥骨骼再生、造血支持及免疫调节功能所需分化潜能的核心前提。目前虽已有诸多针对骨髓来源MSCs（BM-MSCs）及骨相关细胞来源MSCs（BAC-MSCs）的分离方案发表，但现有方法均无法实现无巨噬细胞污染的高纯度MSCs分离，且多数研究未关注这两种来源MSCs的异质性。为获得高纯度的MSCs分离产物，本研究建立了全新策略：通过靶向F4/80/Ly6C/CD45分子，清除巨噬细胞及其前体细胞。通过对两种来源的MSCs在采用或未采用该纯化策略下的转录谱进行分析，本研究证实了不同来源MSCs具有独特的功能特征，同时揭示了巨噬细胞互作对MSCs转录谱的功能性影响。
整体实验设计：本研究共从两种不同组织来源中收集4个细胞群体：骨髓来源（BM）及骨相关细胞来源（BAC）。骨髓来源间充质细胞的获取：从双侧后肢及骨盆的全骨髓中分离得到骨髓来源间充质细胞，接种于Ⅰ型胶原包被的培养板中。初代传代后，一部分样本直接用于后续实验（BM-nonS）；另一部分通过分选获得高纯度的CD31⁻/CD45⁻/Ly6C⁻/F4/80⁻/CD51⁺/Sca-1⁺细胞（BM-S），用于批量RNA测序（bulk RNAseq）。骨相关细胞来源MSCs的获取：从双侧后肢经Ⅰ型胶原酶消化的骨碎片中分离得到BAC来源MSCs。收获细胞后，一部分未经耗竭处理直接用于实验（BAC-nonD）；另一部分通过磁珠分选法去除CD45⁺/Ly6C⁺/F4/80⁺细胞（BAC-D），随后用于批量RNA测序。