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 purpos...

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Bibliographic Details
Published in:Adipocyte 2024-12, Vol.13 (1), p.2350751
Main Authors: Siddiqui, Iram Fatima S., Muthu, Muthu L., Reinhardt, Dieter P.
Format: Article
Language:English
Subjects:
Adipocytes - cytology
Adipocytes - metabolism
Adipogenesis
adipogenic differentiation
adipose tissue
Adipose Tissue - cytology
Adipose Tissue - metabolism
Animals
Bone marrow
Bone Marrow Cells - cytology
Bone Marrow Cells - metabolism
Cell Differentiation
Cell Separation - methods
Cells, Cultured
experimental protocols
Macrophages - cytology
Macrophages - metabolism
mesenchymal stem cells
Mesenchymal Stem Cells - cytology
Mesenchymal Stem Cells - metabolism
Method
Mice
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Summary: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.
ISSN:2162-3945
2162-397X
2162-397X
DOI:10.1080/21623945.2024.2350751