Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein

Abstract

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.

Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II

“…Contrary to the studies above, in which differentiated neurons were investigated, we examined the role of NMII in human neural progenitor cells, a cell population, which plays a pivotal role in the development of nervous system during ontogeny, as well as in neural regeneration. The NPCs applied in our study were committed to the hippocampal dentate gyrus lineage (Yu et al, 2014;Vofely et al, 2018). As dentate gyrus neural progenitor cells are essential for learning, pattern separation, and spatial memory formation, deviations in these cells can cause several disease conditions (Zhao and Overstreet-Wadiche, 2008).…”

“…A human iPSC line, previously generated from fibroblasts of a healthy male individual by Sendai virus reprogramming (Vofely et al, 2018), was kindly provided by Fred H. Gage (Salk Institute), whereas the human ESC line, HUES9 was a kind gift from Douglas A. Melton (Howard Hughes Medical Institute). These cell lines were differentiated into NPCs by a directed differentiation protocol, which is based on a previously published, multistep procedure (Yu et al, 2014), somewhat modified in our laboratory (Supplementary Figure 1).…”

P38 MAPK Promotes Migration and Metastatic Activity of BRAF Mutant Melanoma Cells by Inducing Degradation of PMCA4b

“…Confocal microscopy analysis showed enhanced PMCA4b abundance in the plasma membrane of A375 cells in response to the p38 inhibitor SB202190, and this effect was similar to that seen in the vemurafenib treated cells (Figure 2A). To study if this increase in PMCA4b abundance is reflected in an enhanced Ca 2+ removing capacity of the cells, we performed Ca 2+ signaling measurements using A375 cells stably expressing a genetically encoded green fluorescent Ca 2+ sensor GCAMP6f [23] and confocal imaging. Ca 2+ signaling was initiated by the addition of the Ca 2+ ionophore A23187 (2 µM) that allows Ca 2+ to enter the cells independent of the Ca 2+ channels.…”

“…A375-GFP-PMCA4b and Hela-GFP-PMCA4b stable cell lines were generated by transfection of cells with a SB-CAG-GFP-PMCA4b-CAG-Puromycin construct which contains a Sleeping Beauty transposon system as previously described [17]. For calcium measurement studies, the A375-GCAMP6 stable cell line was generated by transfection with a SB-GCAMP6 construct kindly provided by Prof. Orban laboratory (MTA-TTK) as previously described [23].…”

Functional Comparison of Blood-Derived Human Neural Progenitor Cells

“…The clumps on MEF were further cultured and cloned until stable iPSC lines were generated, whereas the cells on Matrigel were passaged to a poly-ornithine/laminin-coated surface by accutase and cultured in NPC media until homogeneous NPC lines (shortcut NPCs-sNPCs) were established after a few passages. Finally, the donor-derived iPSCs were differentiated into hippocampus-patterned NPCs (classical NPCs-cNPCs) as described previously [22,23] (Figure S1a). We successfully generated proliferative NPCs from all the four donors using two different techniques, and next we started to characterize the sNPCs (made by interrupted reprogramming) and the cNPCs (derived from iPSCs).…”