Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.

Numerous studies have documented the mechanisms that regulate intracellular pH (pHi) in hippocampal neurons in response to an acid load. Here, we studied the response of pHi to depolarization in cultured hippocampal neurons. Elevation of external K+ (6–30 mM) elicited an acid transient followed by a large net alkaline shift. Similar responses were observed in acutely dissociated hippocampal neurons. In Ca2+ free media, the acid response was curtailed and the alkaline shift enhanced. DIDS blocked the alkaline response and revealed a prolonged underlying acidification that was highly dependent on Ca2+ entry. Similar alkaline responses could be elicited by AMPA, indicating that this rise in pHi was a depolarization-induced alkalinization (DIA). The DIA was found to consist of Cl−-dependent and Cl−-independent components, each accounting for about half the peak amplitude. The Cl−-independent component was postulated to arise from operation of the electrogenic Na+-HCO3− cotransporter NBCe1. QPCR and single cell multiplex RT-PCR demonstrated message for NBCe1 in our hippocampal neurons. In neurons cultured from Slc4a4 knockout (KO) mice, the DIA was reduced by roughly half compared with wild type, suggesting that NBCe1 was responsible for the Cl−-independent DIA. In Slc4a4 KO neurons, the remaining DIA was virtually abolished in Cl−-free media. These data demonstrate that DIA of hippocampal neurons occurs via NBCe1, and a parallel DIDS-sensitive, Cl−-dependent mechanism. Our results indicate that by activating net acid extrusion in response to depolarization, hippocampal neurons can preempt a large, prolonged, Ca2+-dependent acidosis.

Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRSs) for schizophrenia (SCZ) and a clinical diagnosis of SCZ, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures in the patient-derived neurons that implicated altered Na+ channel function, action potential interspike interval, and gamma-aminobutyric acid–ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these SCZ patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness is a potentially critical step in generating leads for novel therapeutics.

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across nine time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells that were validated using electrophysiology. We identified widespread changes in the expression of individual transcript features and their splice variants, gene networks, and global patterns of transcription. We next demonstrated that co-culturing human NPCs with rodent astrocytes resulted in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without potentially disruptive cell sorting. We lastly developed and validated a computational tool to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue, which were maturationally heterogeneous but contained subsets of cells most akin to adult human neurons.

In the hippocampus, extracellular carbonic anhydrase (Car) speeds the buffering of an activity-generated rise in extracellular pH that impacts H+-sensitive N-methyl-D-aspartate receptors (NMDARs). We studied the role of Car14 in this brain structure, where it is expressed solely on neurons. Current-clamp responses were recorded from CA1 pyramidal neurons in wild type (WT) vs. Car14 knock out (KO) mice 2 s. before (control) and after (test) a 10 pulse, 100 Hz afferent train. In both WT and KO, the half width (HW) of the test response, and its number of spikes, were augmented relative to the control. An increase in presynaptic release was not involved, as AMPAR-mediated EPSCs were depressed after a train. The increases in HW and spike number were both greater in the Car14 KO. In 0 Mg2+ saline with picrotoxin (using a 20 Hz train), the HW measures were still greater in the KO. The Car inhibitor benzolamide (BZ) enhanced the test response HW in the WT, but had no effect on the already-prolonged HW in the KO. With intracellular MK-801, the curtailed WT and KO responses were indistinguishable, and BZ caused no change. By contrast, the extracellular alkaline changes evoked by the train were not different between WT and KO, and BZ amplified these alkalinizations similarly. These data suggest that Car14 regulates pH transients in the perisynaptic microenvironment, and govern their impact on NMDARs, but plays little role in buffering pH shifts in the broader, macroscopic, extracellular space.