Abstract
Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media, modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells.
Highlights
Bone marrow contains a range of hematopoietic and mesenchymal cell types, including stem cell populations that can be isolated and differentiated in vitro
To test the combined labelling approach, live cultures were incubated with TC, BD and HT before observation under a fluorescence microscope, which revealed strong differences between SM and differentiation medium (DM)
A combined labelling approach was developed to enable lineages found in bone marrow [10]
Summary
Bone marrow contains a range of hematopoietic and mesenchymal cell types, including stem cell populations that can be isolated and differentiated in vitro. Mesenchymal stem cells (MSCs) are multipotent progenitors able to generate bone, adipose and cartilage cell types. MSCs can be induced to form osteoblasts and chondrocytes, two cell types of biomedical importance for strategies aiming to address skeletal damage [1,2,3]. MSCs have been used to generate adipocytes, providing an efficient in vitro model to characterize factors regulating adipose tissue differentiation [4,5]. Since MSCs can form both osteogenic and adipogenic lineages, they represent an important resource for studying the regulation of bone versus adipose cell formation, which can be modelled in vitro [11,12]. Osteogenic differentiation is characterised by the accumulation of mineral deposits in the extracellular matrix, which can be analysed by Alizarin
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