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

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

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Introduction and Purpose 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 door mat 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.Methods and Results 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.Conclusion This laboratory protocol was designed Cord Trousers as a step-by-step procedure for harvesting murine BM-MSCs and ASCs and differentiating them into adipocytes.