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

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.

Development and characterization of a novel, megakaryocyte NF-κB reporter cell line for investigating inflammatory responses.

Essentials An easily detectable readout in megakaryocyte cell lines will enhance inflammatory research in these cells. Here, we report the development and characterization of a novel megakaryocyte NF-κB-reporter cell line (Meg-01R). Multiple inflammatory molecules modulate NF-κB activity in Meg-01R cells. Meg-01R cells respond to small molecule inhibitors such as IMD0354 and C87 that are known to inhibit NF-κB activity upon stimulation with TNFα. ABSTRACT: Background Because of the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. Objectives The primary objective of this study is to develop and functionally characterize a novel megakaryocyte nuclear factor κB (NF-κB) reporter cell line to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. Methods A Meg-01-NF-κB-GFP-Luc (Meg-01R) cell line was developed by inserting a reporter NF-κB-GFP-Luc cassette into normal Meg-01 cells to produce luciferase following activation of NF-κB to enable easy detection of pro-inflammatory and reparative signalling. Results and conclusions Meg-01 and Meg-01R cells have comparable characteristics, including the expression of both GPIbα and integrin ß3 . Meg-01R cells responded to various inflammatory molecules as measured by NF-κB-dependent bioluminescence. For example, inflammatory molecules such as tumor necrosis factor-α and Pam3CSK4 increased NF-κB activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg-01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg-01R cells were able to respond to lipopolysaccharide (LPS; non-ultrapure), although it was not able to react to ultrapure LPS because of the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterization of a novel megakaryocyte NF-κB reporter cell line (Meg-01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF-κB activity.