Time-Dependent Reduction of Calcium Oscillations in Adipose-Derived Stem Cells Differentiating towards Adipogenic and Osteogenic Lineage

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.

An Intracellular Metabolic Signature as a Potential Donor-Independent Marker of the Osteogenic Differentiation of Adipose Tissue Mesenchymal Stem Cells

“…This seems to reflect a more extensive deposition of calcium phosphate nodules for donor 1, consistently with the higher osteogenesis extension shown by ALP levels. Indeed, an increase in total calcium content (including insoluble forms at pH 7) has been reported for hAMSCs undergoing osteogenic differentiation [ 58 ], while calcium stagnation has been interpreted as reflecting stable intracellular calcium levels [ 60 ], thus corroborating the lower mineralization extension characterizing donor 2.…”

“…Total calcium quantification ( Figure S1D ) informs on (i) intracellular calcium, (ii) free calcium ions in the extracellular matrix (ECM), and (iii) calcium ions associated with insoluble mineral deposition (e.g., calcium phosphates). The normalized overall calcium content profile ( Figure S1D ) showed relatively higher values at day 7 for basal conditions for both donors, possibly reflecting high intracellular calcium content [ 60 ]. While calcium levels decreased in basal conditions throughout days 7 to 21 for both donors, in osteogenic conditions a significant increase in calcium content was observed for donor 1 at day 21, while a smaller non-significant increase was noted for donor 2.…”

Store-Operated Ca2+ Entry as a Putative Target of Flecainide for the Treatment of Arrhythmogenic Cardiomyopathy

“…In accordance with this hypothesis, flecainide decreased the frequency of Ca 2+ sparks and normalised the Ca 2+ transients elicited by membrane depolarization in inducible pluripotent stem cell-derived cardiomyocytes isolated from an ACM patient bearing a mutation in the DSC2 gene [ 49 ]. It has long been known that oscillations in intracellular Ca 2+ concentration ([Ca 2+ ] i ) regulate MSC differentiation into a variety of cells belonging to multiple lineages [ 50 , 51 , 52 ]. Moreover, a recent study carried out in our laboratory showed that intracellular Ca 2+ oscillations drive proliferation in C-hMSCs [ 53 ].…”

“…Spontaneous intracellular Ca 2+ oscillations have been described not only in BM-hMSCs [ 54 , 83 , 84 ] but also also in human adipose tissue-derived MSCs (AD-hMSCs) [ 51 , 85 ]. Intracellular Ca 2+ oscillations regulate stem cell differentiation by stimulating the expression of genes that control cellular fate and silencing those that are required for self-renewal [ 52 , 86 , 87 , 88 , 89 , 90 ].…”

Recent Advances in Translational Adipose-Derived Stem Cell Biology

“…In their study, Torre at al. showed that undifferentiated ASCs and cells that differentiate towards the osteogenic and adipogenic fates are characterised by very distinct calcium oscillation patterns [15]. Notably, this methodology can be exploited as a non-invasive technique for assessing ASC differentiation in vitro.…”