Abstract
Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca2+ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.
Highlights
The in vitro generation of neural cells from embryonic stem (ES) cells promises to produce an almost unlimited supply of cells suitable for cell-based replacement therapies in the nervous system [1,2,3,4,5]
The activity of alkaline phosphatase in ES cells was not affected by two pore channel 2 (TPC2) knockdown or overexpression
Manipulation of the TPC2 signaling had little effect on cell proliferation rates and cell cycle profiles of mouse ES cells (Figure 3). These results indicate that the stemness and self-renewal of mouse ES cells are not reliant on TPC2 signaling pathway
Summary
The in vitro generation of neural cells from ES cells promises to produce an almost unlimited supply of cells suitable for cell-based replacement therapies in the nervous system [1,2,3,4,5]. A simple and efficient way to induce ES cells into neural precursors and subsequently generate neuronal and glia cells is to culture ES cells in an adherent monolayer in defined medium [1,2]. In this method, ES cells are cultured in defined serum-free and feeder-free conditions, in the absence of bone morphogenetic protein (BMP) and Wnts signals. ES cells are cultured in defined serum-free and feeder-free conditions, in the absence of bone morphogenetic protein (BMP) and Wnts signals In these conditions, ES cells undergo neural commitment through an autocrine fibroblast growth factor (FGF) signaling mechanism. To more efficiently induce neural commitment of ES cells, it is essential to define novel cellular and molecular events involved in neural differentiation
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