Ultra-Sharp Nanowire Arrays Natively Permeate, Record, and Stimulate Intracellular Activity in Neuronal and Cardiac Networks.

We report innovative scalable, vertical, ultra-sharp nanowire arrays that are individually addressable to enable long-term, native recordings of intracellular potentials. Stable amplitudes of intracellular potentials from 3D tissue-like networks of neurons and cardiomyocytes are obtained. Individual electrical addressability is necessary for high-fidelity intracellular electrophysiological recordings. This study paves the way toward predictive, high-throughput, and low-cost electrophysiological drug screening platforms.

High Density Individually Addressable Nanowire Arrays Record Intracellular Activity from Primary Rodent and Human Stem Cell Derived Neurons.

We report a new hybrid integration scheme that offers for the first time a nanowire-on-lead approach, which enables independent electrical addressability, is scalable, and has superior spatial resolution in vertical nanowire arrays. The fabrication of these nanowire arrays is demonstrated to be scalable down to submicrometer site-to-site spacing and can be combined with standard integrated circuit fabrication technologies. We utilize these arrays to perform electrophysiological recordings from mouse and rat primary neurons and human induced pluripotent stem cell (hiPSC)-derived neurons, which revealed high signal-to-noise ratios and sensitivity to subthreshold postsynaptic potentials (PSPs). We measured electrical activity from rodent neurons from 8 days in vitro (DIV) to 14 DIV and from hiPSC-derived neurons at 6 weeks in vitro post culture with signal amplitudes up to 99 mV. Overall, our platform paves the way for longitudinal electrophysiological experiments on synaptic activity in human iPSC based disease models of neuronal networks, critical for understanding the mechanisms of neurological diseases and for developing drugs to treat them.

Characterization of a subpopulation of developing cortical interneurons from human iPSCs within serum-free embryoid bodies.

Production and isolation of forebrain interneuron progenitors are essential for understanding cortical development and developing cell-based therapies for developmental and neurodegenerative disorders. We demonstrate production of a population of putative calretinin-positive bipolar interneurons that express markers consistent with caudal ganglionic eminence identities. Using serum-free embryoid bodies (SFEBs) generated from human inducible pluripotent stem cells (iPSCs), we demonstrate that these interneuron progenitors exhibit morphological, immunocytochemical, and electrophysiological hallmarks of developing cortical interneurons. Finally, we develop a fluorescence-activated cell-sorting strategy to isolate interneuron progenitors from SFEBs to allow development of a purified population of these cells. Identification of this critical neuronal cell type within iPSC-derived SFEBs is an important and novel step in describing cortical development in this iPSC preparation.

Low-Power Fully Integrated 256-Channel Nanowire Electrode-on-Chip Neural Interface for Intracellular Electrophysiology.

Intracellular electrophysiology, a vital and versatile technique in cellular neuroscience, is typically conducted using the patch-clamp method. Despite its effectiveness, this method poses challenges due to its complexity and low throughput. The pursuit of multi-channel parallel neural intracellular recording has been a long-standing goal, yet achieving reliable and consistent scaling has been elusive because of several technological barriers. In this work, we introduce a micropower integrated circuit, optimized for scalable, high-throughput in vitro intrinsically intracellular electrophysiology. This system is capable of simultaneous recording and stimulation, implementing all essential functions such as signal amplification, acquisition, and control, with a direct interface to electrodes integrated on the chip. The electrophysiology system-on-chip (eSoC), fabricated in 180nm CMOS, measures 2.236 mm × 2.236 mm. It contains four 8 × 8 arrays of nanowire electrodes, each with a 50 µm pitch, placed over the top-metal layer on the chip surface, totaling 256 channels. Each channel has a power consumption of 0.47 µW, suitable for current stimulation and voltage recording, and covers 80 dB adjustable range at a sampling rate of 25 kHz. Experimental recordings with the eSoC from cultured neurons in vitro validate its functionality in accurately resolving chemically induced multi-unit intracellular electrical activity.

Assaying Chemical Long-Term Potentiation in Human iPSC-Derived Neuronal Networks.

Impairment of long-term potentiation (LTP) is a common feature of many preclinical models of neurological disorders. Modeling LTP on human induced pluripotent stem cells (hiPSC) enables the investigation of this critical plasticity process in disease-specific genetic backgrounds. Here, we describe a method to chemically induce LTP across entire networks of hiPSC-derived neurons on multi-electrode arrays (MEAs) and investigate effects on neuronal network activity and associated molecular changes.

Development of a platform to investigate long-term potentiation in human iPSC-derived neuronal networks.

Impairment of long-term potentiation (LTP) is a common feature of many pre-clinical models of neurological disorders; however, studies in humans are limited by the inaccessibility of the brain. Human induced pluripotent stem cells (hiPSCs) provide a unique opportunity to study LTP in disease-specific genetic backgrounds. Here we describe a multi-electrode array (MEA)-based assay to investigate chemically induced LTP (cLTP) across entire networks of hiPSC-derived midbrain dopaminergic (DA) and cortical neuronal populations that lasts for days, allowing studies of the late phases of LTP and enabling detection of associated molecular changes. We show that cLTP on midbrain DA neuronal networks is largely independent of the N-methyl-D-aspartate receptor (NMDAR) and partially dependent on brain-derived neurotrophic factor (BDNF). Finally, we describe activity-regulated gene expression induced by cLTP. This cLTP-MEA assay platform will enable phenotype discovery and higher-throughput analyses of synaptic plasticity on hiPSC-derived neurons.

Super-Selective Reconstruction of Causal and Direct Connectivity With Application to in vitro iPSC Neuronal Networks.

Despite advancements in the development of cell-based in-vitro neuronal network models, the lack of appropriate computational tools limits their analyses. Methods aimed at deciphering the effective connections between neurons from extracellular spike recordings would increase utility of in vitro local neural circuits, especially for studies of human neural development and disease based on induced pluripotent stem cells (hiPSC). Current techniques allow statistical inference of functional couplings in the network but are fundamentally unable to correctly identify indirect and apparent connections between neurons, generating redundant maps with limited ability to model the causal dynamics of the network. In this paper, we describe a novel mathematically rigorous, model-free method to map effective-direct and causal-connectivity of neuronal networks from multi-electrode array data. The inference algorithm uses a combination of statistical and deterministic indicators which, first, enables identification of all existing functional links in the network and then reconstructs the directed and causal connection diagram via a super-selective rule enabling highly accurate classification of direct, indirect, and apparent links. Our method can be generally applied to the functional characterization of any in vitro neuronal networks. Here, we show that, given its accuracy, it can offer important insights into the functional development of in vitro hiPSC-derived neuronal cultures.

Connectivity and circuitry in a dish versus in a brain.

In order to understand and find therapeutic strategies for neurological disorders, disease models that recapitulate the connectivity and circuitry of patients' brain are needed. Owing to many limitations of animal disease models, in vitro neuronal models using patient-derived stem cells are currently being developed. However, prior to employing neurons as a model in a dish, they need to be evaluated for their electrophysiological properties, including both passive and active membrane properties, dynamics of neurotransmitter release, and capacity to undergo synaptic plasticity. In this review, we survey recent attempts to study these issues in human induced pluripotent stem cell-derived neurons. Although progress has been made, there are still many hurdles to overcome before human induced pluripotent stem cell-derived neurons can fully recapitulate all of the above physiological properties of adult mature neurons. Moreover, proper integration of neurons into pre-existing circuitry still needs to be achieved. Nevertheless, in vitro neuronal stem cell-derived models hold great promise for clinical application in neurological diseases in the future.

Low-amplitude high frequency vibration down-regulates myostatin and atrogin-1 expression, two components of the atrophy pathway in muscle cells.

Whole body vibration (WBV) is a very widespread mechanical stimulus used in physical therapy, rehabilitation and fitness centres. It has been demonstrated that vibration induces improvements in muscular strength and performance and increases bone density. We investigated the effects of low-amplitude, high frequency vibration (HFV) at the cellular and tissue levels in muscle. We developed a system to produce vibrations adapted to test several parameters in vitro and in vivo. For in vivo experiments, we used newborn CD1 wild-type mice, for in vitro experiments, we isolated satellite cells from 6-day-old CD1 mice, while for proliferation studies, we used murine cell lines. Animals and cells were treated with high frequency vibration at 30 Hz. We analyzed the effects of mechanical stimulation on muscle hypertrophy/atrophy pathways, fusion enhancement of myoblast cells and modifications in the proliferation rate of cells. Results demonstrated that mechanical vibration strongly down-regulates atrophy genes both in vivo and in vitro. The in vitro experiments indicated that mechanical stimulation promotes fusion of satellite cells treated directly in culture compared to controls. Finally, proliferation experiments indicated that stimulated cells had a decreased growth rate compared to controls. We concluded that vibration treatment at 30 Hz is effective in suppressing the atrophy pathway both in vivo and in vitro and enhances fusion of satellite muscle cells.

A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs).

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48-55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.

Characterization and molecular profiling of PSEN1 familial Alzheimer's disease iPSC-derived neural progenitors.

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aß42 to Aß40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aß42/40, and have characterized novel expression changes.

Effects of low-amplitude, high-frequency vibrations on proliferation and differentiation of SAOS-2 human osteogenic cell line.

The aim of the work was to understand the consequences of low-amplitude, high-frequency vibrations on proliferation and differentiation of SAOS-2 cells (sarcoma osteogenetic), an osteoblastic and tumorigenic cell line. We realized a bioreactor composed of an eccentric motor that produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. The cultures of SAOS-2 cells were fixed on the plate, and the linear acceleration provoked by the motor to the cultures was measured. We used 30 Hz as stimulating frequency after a preliminary test on the effect of different frequencies on differentiation of cells. Afterward, SAOS-2 cells were stimulated with 30 Hz for different durations, every day for 4 days. The expression of some genes involved in the differentiation process was analyzed first with a reverse transcriptase-polymerase chain reaction and afterward with a real-time polymerase chain reaction on the most expressed genes. Moreover, the proliferation of cells was evaluated. The results suggest a strong increase in the expression of the genes involved in tissue differentiation in the treated groups with respect to the controls. On the other hand, the proliferation seems to be slowed down, so probably the acceleration perceived by the mechanosensors of the cells changes the cellular cycle by blocking the duplication to early differentiate toward bone tissue.