Abstract
Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval—IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.
Highlights
Mesenchymal stem cells (MSCs) are multipotent, fibroblast-like cells that can be readily obtained from various adult tissues including the bone marrow, adipose tissue, and peripheral blood as well as from prenatal tissues such as amniotic fluid, umbilical cord, and placenta
We confirmed that Adipose-derived stem cells (ASCs) treated with osteogenic differentiation medium showed extracellular, insoluble deposits of CaPO4 as shown by Alizarin RedS staining at 21 days in vitro (DIV)
Using 2-photon microscopy, we have identified fluorescent deposits, which likely correspond to deposits of CaPO4, early as 7 DIV (Figure 1C, bottom panels: note the extracellular, fluorescent deposits)
Summary
Mesenchymal stem cells (MSCs) are multipotent, fibroblast-like cells that can be readily obtained from various adult tissues including the bone marrow, adipose tissue, and peripheral blood as well as from prenatal tissues such as amniotic fluid, umbilical cord, and placenta. As they are derived from the mesoderm, their differentiation spectrum is limited to mesenchymal derivatives such as bone, fat, and cartilage Adipose-derived stem cells (ASCs) are tissue-resident ASCs that can be isolated from lipo-aspirates [11] Their differentiation potential towards the mesenchymal fate is comparable to that of ASCs from other organs including the bone marrow [12,13]. Like bone marrow ASCs, they are able to contribute to regeneration of various tissues via paracrine effects [13,14,15]
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