Abstract
The in vitro use of neurons that are differentiated from human induced pluripotent stem cells (hiPSC-neurons) is expected to improve the prediction accuracy of preclinical tests for both screening and safety assessments in drug development. To achieve this goal, hiPSC neurons are required to differentiate into functional neurons that form excitatory networks and stably express N-methyl-D-aspartate receptors (NMDARs). Recent studies have identified some astrocyte-derived factors that are important for the functional maturation of neurons. We therefore examined the effects of the astrocyte-derived factor glypican 6 (GPC6) on hiPSC-neurons. When we pharmacologically examined which receptor subtypes mediate L-glutamate (L-Glu)-induced changes in the intracellular Ca2+ concentrations in hiPSC neurons using fura-2 Ca2+ imaging, NMDAR-mediated responses were not detected through 7 days in vitro (DIV). These cells were also not vulnerable to excitotoxicity at 7 DIV. However, a 5-days treatment with GPC6 from 3 DIV induced an NMDAR-mediated Ca2+ increase in hiPSC-neurons and increased the level of NMDARs on the cell surface. We also found that GPC6-treated hiPSC-neurons became responsive to excitotoxicity. These results suggest that GPC6 increases the level of functional NMDARs in hiPSC-neurons. Glial factors may play a key role in accelerating the functional maturation of hiPSC neurons for drug-development applications.
Highlights
Cellular reprogramming, in which terminally differentiated somatic cells are converted into pluripotent stem cells (Takahashi et al, 2007) is clinically relevant and useful in drug development because these cells allow for the homogeneous derivation of mature human cell types in large quantities without the ethical issues associated with embryonic stem cells
We examined the expression of the neuronal marker β3 tubulin, the stem-cell marker Nestin, and the radial glia/astrocyte marker GFAP at 3 days in vitro (DIV)
We examined the contributions of N-methyl-D-aspartate receptors (NMDARs) and non-NMDARs to the L-Glu-induced Ca2+ increase in hiPSCneurons
Summary
In which terminally differentiated somatic cells are converted into pluripotent stem cells (induced pluripotent stem cells, i.e., hiPSCs) (Takahashi et al, 2007) is clinically relevant and useful in drug development because these cells allow for the homogeneous derivation of mature human cell types in large quantities without the ethical issues associated with embryonic stem cells. Effects of Glypican 6 on hiPSC-Neurons accuracy of the effects of new candidate compounds in humans. To this end, it is required that hiPSC-neurons differentiate to functional neurons that form excitatory networks and stably express N-methyl-D-aspartate receptors (NMDARs). Huge numbers of astrocytes are generated just prior to the postnatal wave of synaptogenesis, and astrocyte-derived factors play important roles in the functional differentiation of neurons in the developing brain (Wang and Bordey, 2008). Glial factors may play a key role in accelerating the functional maturation of hiPSC neurons for drug-development applications
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