Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors

Abstract

The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors-namely,N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)-are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region-specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus-derived neural stem/progenitor cells-namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50= 7.5 ± 0.4 µM; n= 4), AMPA (EC50= 2.5 ± 0.1 µM; n= 3), or KA (EC50= 33.5 ± 1.1 µM; n= 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor-namely, MK-801 for NMDA stimulation (IC50= 0.6 ± 0.1 µM; n= 4) and NBQX for AMPA and KA stimulation (IC50= 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n= 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.

Evolutionary origin of Tbr2‐expressing precursor cells and the subventricular zone in the developing cortex

“…Western blot analysis showed that labels a band of 47-50 kD, and it was characterized by using immunohistochemistry in mouse (Stevens et al, 2010). Our staining with this antibody matched that from original descriptions of Pax6 immunostaining in the developing cortex (Gotz et al, 1998), our previous publications (Martinez-Cerdeno et al, 2012; Cunningham et al, 2013a), and more recent work from unrelated laboratories (Fukushima et al, 2014; Maury et al, 2015). The database link for PAX6 in Homo sapiens (human) .…”

Ketamine Increases Proliferation of Human iPSC-Derived Neuronal Progenitor Cells via Insulin-Like Growth Factor 2 and Independent of the NMDA Receptor

“…Here, we identified proliferative effects and first hints towards the underlying molecular mechanism of the rapid-acting and long-lasting antidepressant ketamine in human NPCs. The undifferentiated human iPSC-derived NPCs used in our study displayed typical characteristics of hippocampal NPCs by upregulating neuronal progenitor markers such Nestin and Pax6 while missing expression of functional ionotropic glutamate receptors, such as NMDA or AMPA receptors, which is in accordance with the previously described characterization of hippocampal human undifferentiated NPCs [9,28,39].…”

“…Cells were seeded at a density of 3.0 × 10 5 cells per ml and incubated for 24 h. The following day, the medium was removed and cells were loaded with 4 µM Fluo-8 (AAT Bioquest) as the indicator dye, reconstituted in a modified Tyrode’s assay buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 135 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , and 10 mM glucose adjusted to pH = 7.4.) and incubated for 30 min at 37 °C and 5% CO 2 , and another 30 min at room temperature in the dark [27,28]. Then, the Fluo-8 loading buffer was removed and replaced with fresh Tyrode’s assay buffer.…”

Establishment of a Human Neuronal Network Assessment System by Using a Human Neuron/Astrocyte Co-Culture Derived from Fetal Neural Stem/Progenitor Cells

“…Before neural differentiation, expanded HIP-009 cells were harvested and cultured in Neural Transition Medium (PhoenixSongs Biologicals) added with transition medium supplement cocktail (Neural Transition Supplement, 10 ng/mL bFGF, 20 ng/mL EGF, and 1 µg/mL laminin) and 30 µg/mL gentamicin for 3 days. 14 The expansion and transition treatment of HIP-009 cells was carried out at 37 °C in a humidified atmosphere of 2% O 2 , balanced with N 2 and 6% CO 2 .…”