In vitro differentiation of W8B2+ human cardiac stem cells: gene expression of ionic channels and spontaneous calcium activity.

BACKGROUND: Human cardiac stem cells expressing the W8B2 marker (W8B2+ CSCs) were recently identified and proposed as a new model of multipotent CSCs capable of differentiating into smooth muscle cells, endothelial cells and immature myocytes. Nevertheless, no characterization of ion channel or calcium activity during the differentiation of these stem cells has been reported. METHODS: The objectives of this study were thus to analyze (using the TaqMan Low-Density Array technique) the gene profile of W8B2+ CSCs pertaining to the regulation of ion channels, transporters and other players involved in the calcium homeostasis of these cells. We also analyzed spontaneous calcium activity (via the GCaMP calcium probe) during the in vitro differentiation of W8B2+ CSCs into cardiac myocytes. RESULTS: Our results show an entirely different electrophysiological genomic profile between W8B2+ CSCs before and after differentiation. Some specific nodal genes, such as Tbx3, HCN, ICaT, L, KV, and NCX, are overexpressed after this differentiation. In addition, we reveal spontaneous calcium activity or a calcium clock whose kinetics change during the differentiation process. A pharmacological study carried out on differentiated W8B2+ CSCs showed that the NCX exchanger and IP3 stores play a fundamental role in the generation of these calcium oscillations. CONCLUSIONS: Taken together, the present results provide important information on ion channel expression and intrinsic calcium dynamics during the differentiation process of stem cells expressing the W8B2 marker.

Functional BKCa channel in human resident cardiac stem cells expressing W8B2.

Recently, a new population of resident cardiac stem cells (CSCs) positive for the W8B2 marker has been identified. These CSCs are considered to be an ideal cellular source to repair myocardial damage after infarction. However, the electrophysiological profile of these cells has not been characterized yet. We first establish the conditions of isolation and expansion of W8B2+ CSCs from human heart biopsies using a magnetic sorting system followed by flow cytometry cell sorting. These cells display a spindle-shaped morphology, are highly proliferative, and possess self-renewal capacity demonstrated by their ability to form colonies. Besides, W8B2+ CSCs are positive for mesenchymal markers but negative for hematopoietic and endothelial ones. RT-qPCR and immunostaining experiments show that W8B2+ CSCs express some early cardiac-specific transcription factors but lack the expression of cardiac-specific structural genes. Using patch clamp in the whole-cell configuration, we show for the first time the electrophysiological signature of BKCa current in these cells. Accordingly, RT-PCR and western blotting analysis confirmed the presence of BKCa at both mRNA and protein levels in W8B2+ CSCs. Interestingly, BKCa channel inhibition by paxilline decreased cell proliferation in a concentration-dependent manner and halted cell cycle progression at the G0/G1 phase. The inhibition of BKCa also decreased the self-renewal capacity but did not affect migration of W8B2+ CSCs. Taken together, our results are consistent with an important role of BKCa channels in cell cycle progression and self-renewal in human cardiac stem cells.

Optogenetic approach for targeted activation of global calcium transients in differentiated C2C12 myotubes.

Excitation-contraction coupling in muscle cells is initiated by a restricted membrane depolarization delimited within the neuromuscular junction. This targeted depolarization triggers an action potential that propagates and induces a global cellular calcium response and a consequent contraction. To date, numerous studies have investigated this excitation-calcium response coupling by using different techniques to depolarize muscle cells. However, none of these techniques mimic the temporal and spatial resolution of membrane depolarization observed in the neuromuscular junction. By using optogenetics in C2C12 muscle cells, we developed a technique to study the calcium response following membrane depolarization induced by photostimulations of membrane surface similar or narrower than the neuromuscular junction area. These stimulations coupled to confocal calcium imaging generate a global cellular calcium response that is the consequence of a membrane depolarization propagation. In this context, this technique provides an interesting, contactless and relatively easy way of investigation of calcium increase/release as well as calcium decrease/re-uptake triggered by a propagated membrane depolarization.

Antibiotics inhibit sphere-forming ability in suspension culture.

BACKGROUND: This last decade, a lot of emphasis has been placed on developing new cancer cell culture models, closer to in vivo condition, in order to test new drugs and therapies. In the case of colorectal cancer, the use of patient biopsies to seed 3D primary cultures and mimic tumor initiation necessitates the use of antibiotics to prevent microbial intestinal contamination. However, not only long term use of antibiotics may mask the presence of low levels of microbial contamination, it may also impact cancer cell phenotype. METHODS: In this study we tested the impact of penicillin-streptomycin cocktail addition in both monolayer and suspension culture. To ensure the reliability of our observations we used six different cell lines and each experiment was performed in triplicate. Results were analyzed with Student's t test. RESULTS: We show that penicillin-streptomycin cocktail inhibits the sphere-forming ability of six cancer cell lines in suspension culture though it has no impact in monolayer culture. We correlate this effect with a significant decrease of cancer stem cells pool which holds self-renewal potential. CONCLUSIONS: Overall, this study warns against systematic addition of antibiotics in growth medium and raises the interesting possibility of using antibiotics to target cancer stem cells.