Tissue Engineering and Regenerative Medicine

Human pluripotent stem cells (hPSC) provide new approaches to study the development and differentiation of various cell types of the human body and research tools for disease modeling. Meanwhile, the basic signaling events, including Ca2+-signals, have not been comprehensively evaluated in these cells. Therefore, we have started to study intracellular calcium responses to various ligands and the calcium signaling pathways in hPSCs and in their differentiated derivatives. First, we developed a protocol using the small molecule fluorescent Ca2+-indicator Fluo-4 and confocal microscopy. While this method was informative, several technical problems hindered this approach. To overcome these difficulties (i.e. dye-loading and toxicity), we generated hPSC lines which stably express a genetically encoded Ca2+-indicator (GCaMP2) protein by using a transposon-based gene delivery method. In this stable indicator system, we successfully studied the effects of various ligands in undifferentiated hPSCs and in hPSC-derived cardiomyocytes. To extend our knowledge towards Ca2+-signals in hPSC-derived neural cell types, we also generated GCaMP6 fast expressing neural progenitor cells (NPCs) and then differentiated these to PROX1-positive hippocampal neurons. Next, we investigated the calcium signals in NPCs and PROX1-positive hippocampal neurons expressing GCaMP6 fast, or loaded with Fluo-4 in parallel experiments. We found that specific, ligand induced Ca2+ signals were similar in the two assays, suggesting the applicability of GCaMP6f for the investigation of fast Ca2+ signals in neural cultures. We conclude that characteristics of both the spontaneous and ligand-induced Ca2+-signals, as well as their pharmacological modifications can be successfully examined in these model cells by fluorescence imaging.