Caffeine-induced Ca2+ signaling as an index of cardiac progenitor cells differentiation

Abstract

Cardiac progenitor cells (CPCs), migrating from heart tissue, in culture aggregate to form cardiospheres (CSs) in which replication and cardiogenic differentiation occur. However, the frequency of functional differentiation in CSs and the role of cell clustering in supporting it remain to be established. The aim of our study is to quantify differentiation of a muscle-type Ca(2+) release mechanism in CS-derived cells, correlate it with cardiac differentiation markers and test its dependency on CS formation. CPCs migrating from murine cardiac explants were studied prior and after CSs formation (Pre-CS and Post-CS). Inducibility of RyR- and IP3-R-mediated Ca(2+) transients in individual cells was tested by exposure to caffeine and ATP, respectively; expression of cardiac and non-cardiac lineage markers was assessed. Caffeine responsiveness was negligible in Pre-CS cells and increased by 7.5 fold in Post-CS cells (3.6 vs. 26.9%; p < 0.05), and was closely correlated with activation of the cardiac TnI gene promoter. ATP-induced responses, frequent in Pre-CS (86%), were slightly increased in Post-CS cells (94%; p < 0.05). Expression of cardiac-specific Ca(2+)-handling proteins (Cav1.2, NCX1, RyR2, SERCA2a) was either limited to the Post-CS stage, or markedly enhanced. CS beating was infrequent, but its pharmacology was compatible with cardiac excitation-contraction coupling. Expression of non-cardiac lineage was low in general, and similar between Pre- and Post-CS cells. Culture conditions inhibiting CSs formation prevented the increase in caffeine responders. In conclusion, clustering in CSs leads to the induction of a muscle-specific functional response in about 30% of CPCs; this is accompanied by development of a cardiac-specific expression pattern.

Post-natal cardiomyocytes can generate iPS cells with an enhanced capacity toward cardiomyogenic re-differentation

“…Responsiveness to caffeine, 16 which acts on muscle-specific ryanodine receptor channels, was significantly higher in differentiated CM-derived iPS cells than in differentiated CF-derived iPS cells. Responsiveness to nicotine, acting on the nicotinic acetylcholine receptor (nAChR), was negligible in all cell types, and this ruled out differentiation toward skeletal muscle.…”

TGFβ-Dependent Epithelial-to-Mesenchymal Transition Is Required to Generate Cardiospheres from Human Adult Heart Biopsies

“…Interestingly, some of the same genes also changed significantly in CDCs versus EDCs, but in the opposite way compared with 3D structures (SNAI1, KLF4, and NOTCH3), indicating divergence of lineage specification between 3D and monolayer cultures. In some cases, however, gene profiles in CDCs were more similar to CSps than to EDCs (e.g., [TGFBR2, YTHY1, [TEK, and YBMP4), suggesting that CDCs represent an intermediate stage that retains memory of passing through the CSp stage, which seems to function as a selective/ inductive stage [50]). Together, these data indicate that the formation of the 3D structures involves EMT and supports the differentiation of cardiac progenitors.…”

Induced pluripotent stem cells: progress towards a biomedical application

“…Development of such a store can be quantitatively assessed by Ca 2+ -imaging techniques (i.e., by counting the proportion of cells responding with a Ca 2+ transient to challenge with caffeine, a powerful RyR opener). Such a proportion has been shown to increase in parallel with cardiogenic differentiation of murine cardiac precursors [24].…”