Tonyalı Gülsena, Kiliç Emine, Muratoğlu Bihter, Alpdündar-Bulut Esin, Özdemir Cansu, Uçkan-Çetinkaya Duygu
Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Ankara, Turkey.
Institute of Health Sciences, Department of Stem Cell Sciences, Hacettepe University, Ankara, Turkey.
Curr Protoc. 2025 Jan;5(1):e70081. doi: 10.1002/cpz1.70081.
Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM-MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non-enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell & Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human BMAT-MSCs from the fatty layer of the bone marrow Basic Protocol 2: Culture expansion, trypsinization, and cryopreservation of BMAT-MSCs Support Protocol 1: Immunophenoypic characterization of human BMAT-MSCs by flow cytometry Support Protocol 2: In vitro characterization of multilineage differentiation potential of human BMAT-MSCs Support Protocol 3: Further characterization of gene expression in human BMAT-MSCs using qRT-PCR.
骨髓脂肪组织（BMAT）因其在白血病发病机制、癌症转移和骨髓衰竭中的关键作用而备受关注。BMAT是一种代谢活跃的独特组织，与其他脂肪储存部位不同。骨髓脂肪细胞与造血干/祖细胞和成骨细胞密切相互作用，在调节它们的功能中起关键作用。然而，分离和定义人BMAT（hBMAT）的标准化方法仍不明确。在动物模型中，BMAT通常通过冲洗、酶消化或机械破坏直接从骨髓中分离。在人类中，BMAT的分离通常涉及对源自骨髓抽吸物的骨髓间充质干/基质细胞（BM-MSCs）进行成脂诱导。然而，这种方法反映的是细胞对化学刺激的反应，可能无法准确代表体内分化潜能。同样，由于炎症影响，从髋关节或膝关节置换手术中获得的BMAT可能无法反映基础生理状况。在此，我们描述了一种直接从健康移植供体的骨髓抽吸物脂肪层培养BMAT的方法。该方案采用离心和洗涤步骤，使用基本实验室设备，提供了简单且非酶促的方法。为进行验证，根据国际细胞与基因治疗协会（ISCT）标准对分离的细胞进行表征。© 2025威利期刊有限责任公司。基本方案1：从骨髓脂肪层分离人BMAT-MSCs 基本方案2：BMAT-MSCs的培养扩增、胰蛋白酶消化和冷冻保存 支持方案1：通过流式细胞术对人BMAT-MSCs进行免疫表型表征 支持方案2：人BMAT-MSCs多谱系分化潜能的体外表征 支持方案3：使用qRT-PCR对人BMAT-MSCs中的基因表达进行进一步表征。
