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

Abstract

ABSTRACT 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 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 as a step-by-step procedure for harvesting murine BM-MSCs and ASCs and differentiating them into adipocytes.