Establishment of a human induced pluripotent stem cell line (TAUi008-A) derived from a multiple sclerosis patient.

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system where the main pathogenetic events include demyelination and axonal degeneration. Here, we generated a human induced pluripotent stem cell (hiPSC) line from peripheral blood mononuclear cells of an MS patient utilizing Sendai virus reprogramming. The produced hiPSC line expressed pluripotency markers, differentiated into three germ layers, showed a normal karyotype and was free of virus vectors, transgenes and mycoplasma. Established hiPSCs are a valuable source for studies of MS disease modeling and drug discovery.

Comparative microelectrode array data of the functional development of hPSC-derived and rat neuronal networks.

We present a dataset of microelectrode array (MEA) recordings from human pluripotent stem cell (hPSC)-derived and rat embryonic cortical neurons during their in vitro maturation. The data were prepared to assess extracellularly recorded spontaneous activity and to compare the functional development of these neuronal networks. In addition to recordings of spontaneous activity, we provide pharmacological responses of hPSC-derived and rat cortical cultures at their mature stage. Together with the recorded electrode raw data, we share the analysis code to form a comprehensive dataset including spike times, spike waveforms, burst activity and network synchronization metrics calculated with two different connectivity estimators. Moreover, we provide the analysis code that produced the key scientific findings published previously with this dataset. This large dataset enables investigation of the functional aspects of maturing cortical neuronal networks and provides substantial parameters to assess the differences and similarities between hPSC-derived and rat cortical networks in vitro. This publicly available dataset will be beneficial, especially for experimental and computational neuroscientists.

A modular brain-on-a-chip for modelling epileptic seizures with functionally connected human neuronal networks.

Epilepsies are a group of neurological disorders characterised by recurrent epileptic seizures. Seizures, defined as abnormal transient discharges of neuronal activity, can affect the entire brain circuitry or remain more focal in the specific brain regions and neuronal networks. Human pluripotent stem cell (hPSC)-derived neurons are a promising option for modelling epilepsies, but as such, they do not model groups of connected neuronal networks or focal seizures. Our solution is a Modular Platform for Epilepsy Modelling In Vitro (MEMO), a lab-on-chip device, in which three hPSC-derived networks are separated by a novel microfluidic cell culture device that allows controlled network-to-network axonal connections through microtunnels. In this study, we show that the neuronal networks formed a functional circuitry that was successfully cultured in MEMO for up to 98 days. The spontaneous neuronal network activities were monitored with an integrated custom-made microelectrode array (MEA). The networks developed spontaneous burst activity that was synchronous both within and between the axonally connected networks, i.e. mimicking both local and circuitry functionality of the brain. A convulsant, kainic acid, increased bursts only in the specifically treated networks. The activity reduction by an anticonvulsant, phenytoin, was also localised to treated networks. Therefore, modelling focal seizures in human neuronal networks is now possible with the developed chip.

Covalent immobilization of luminescent oxygen indicators reduces cytotoxicity.

Luminescence-based oxygen sensing is a widely used tool in cell culture applications. In a typical configuration, the luminescent oxygen indicators are embedded in a solid, oxygen-permeable matrix in contact with the culture medium. However, in sensitive cell cultures even minimal leaching of the potentially cytotoxic indicators can become an issue. One way to prevent the leaching is to immobilize the indicators covalently into the supporting matrix. In this paper, we report on a method where platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) oxygen indicators are covalently immobilized into a polymer matrix consisting of polystyrene and poly(pentafluorostyrene). We study how the covalent immobilization influences the sensing material's cytotoxicity to human induced pluripotent stem cell-derived (hiPSC-derived) neurons and cardiomyocytes (CMs) through 7-13 days culturing experiments and various viability analyses. Furthermore, we study the effect of the covalent immobilization on the indicator leaching and the oxygen sensing properties of the material. In addition, we demonstrate the use of the covalently linked oxygen sensing material in real time oxygen tension monitoring in functional hypoxia studies of the hiPSC-derived CMs. The results show that the covalently immobilized indicators substantially reduce indicator leaching and the cytotoxicity of the oxygen sensing material, while the influence on the oxygen sensing properties remains small or nonexistent.

Transparent Microelectrode Arrays Fabricated by Ion Beam Assisted Deposition for Neuronal Cell in Vitro Recordings.

Microelectrode array (MEA) is a tool used for recording bioelectric signals from electrically active cells in vitro. In this paper, ion beam assisted electron beam deposition (IBAD) has been used for depositing indium tin oxide (ITO) and titanium nitride (TiN) thin films which are applied as transparent track and electrode materials in MEAs. In the first version, both tracks and electrodes were made of ITO to guarantee full transparency and thus optimal imaging capability. In the second version, very thin (20 nm) ITO electrodes were coated with a thin (40 nm) TiN layer to decrease the impedance of Ø30 µm electrodes to one third (1200 kΩ 320 kΩ) while maintaining (partial) transparency. The third version was also composed of transparent ITO tracks, but the measurement properties were optimized by using thick (200 nm) opaque TiN electrodes. In addition to the impedance, the optical transmission and electric noise levels of all three versions were characterized and the functionality of the MEAs was successfully demonstrated using human pluripotent stem cell-derived neuronal cells. To understand more thoroughly the factors contributing to the impedance, MEAs with higher IBAD ITO thickness as well as commercial sputter-deposited and highly conductive ITO were fabricated for comparison. Even if the sheet-resistance of our IBAD ITO thin films is very high compared to the sputtered one, the impedances of the MEAs of each ITO grade were found to be practically equal (e.g., 300-370 kΩ for Ø30 µm electrodes with 40 nm TiN coating). This implies that the increased resistance of the tracks, either caused by lower thickness or lower conductivity, has hardly any contribution to the impedance of the MEA electrodes. The impedance is almost completely defined by the double-layer interface between the electrode top layer and the medium including cells.

Co-stimulation with IL-1ß and TNF-α induces an inflammatory reactive astrocyte phenotype with neurosupportive characteristics in a human pluripotent stem cell model system.

Astrocyte reactivation has been discovered to be an important contributor to several neurological diseases. In vitro models involving human astrocytes have the potential to reveal disease-specific mechanisms of these cells and to advance research on neuropathological conditions. Here, we induced a reactive phenotype in human induced pluripotent stem cell (hiPSC)-derived astrocytes and studied the inflammatory natures and effects of these cells on human neurons. Astrocytes responded to interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) treatment with a typical transition to polygonal morphology and a shift to an inflammatory phenotype characterized by altered gene and protein expression profiles. Astrocyte-secreted factors did not exert neurotoxic effects, whereas they transiently promoted the functional activity of neurons. Importantly, we engineered a novel microfluidic platform designed for investigating interactions between neuronal axons and reactive astrocytes that also enables the implementation of a controlled inflammatory environment. In this platform, selective stimulation of astrocytes resulted in an inflammatory niche that sustained axonal growth, further suggesting that treatment induces a reactive astrocyte phenotype with neurosupportive characteristics. Our findings show that hiPSC-derived astrocytes are suitable for modeling astrogliosis, and the developed in vitro platform provides promising novel tools for studying neuron-astrocyte crosstalk and human brain disease in a dish.

Functional characterization of human pluripotent stem cell-derived cortical networks differentiated on laminin-521 substrate: comparison to rat cortical cultures.

Human pluripotent stem cell (hPSC)-derived neurons provide exciting opportunities for in vitro modeling of neurological diseases and for advancing drug development and neurotoxicological studies. However, generating electrophysiologically mature neuronal networks from hPSCs has been challenging. Here, we report the differentiation of functionally active hPSC-derived cortical networks on defined laminin-521 substrate. We apply microelectrode array (MEA) measurements to assess network events and compare the activity development of hPSC-derived networks to that of widely used rat embryonic cortical cultures. In both of these networks, activity developed through a similar sequence of stages and time frames; however, the hPSC-derived networks showed unique patterns of bursting activity. The hPSC-derived networks developed synchronous activity, which involved glutamatergic and GABAergic inputs, recapitulating the classical cortical activity also observed in rodent counterparts. Principal component analysis (PCA) based on spike rates, network synchronization and burst features revealed the segregation of hPSC-derived and rat network recordings into different clusters, reflecting the species-specific and maturation state differences between the two networks. Overall, hPSC-derived neural cultures produced with a defined protocol generate cortical type network activity, which validates their applicability as a human-specific model for pharmacological studies and modeling network dysfunctions.

Microelectrode Array With Transparent ALD TiN Electrodes.

Low noise platinum black or sputtered titanium nitride (TiN) microelectrodes are typically used for recording electrical activity of neuronal or cardiac cell cultures. Opaque electrodes and tracks, however, hinder the visibility of the cells when imaged with inverted microscope, which is the standard method of imaging cells plated on microelectrode array (MEA). Even though transparent indium tin oxide (ITO) electrodes exist, they cannot compete in impedance and noise performance with above-mentioned opaque counterparts. In this work, we propose atomic layer deposition (ALD) as the method to deposit TiN electrodes and tracks which are thin enough (25-65 nm) to be transparent (transmission ∼18-45%), but still benefit from the columnar structure of TiN, which is the key element to decrease noise and impedance of the electrodes. For ALD TiN electrodes (diameter 30 µm) impedances from 510 to 590 kΩ were measured at 1 kHz, which is less than the impedance of bare ITO electrodes. Human induced pluripotent stem cell (hiPSC)-derived cortical neurons were cultured on the ALD TiN MEAs for 14 days without observing any biocompatibility issues, and spontaneous electrical activity of the neurons was recorded successfully. The results show that transparent ALD TiN film is a suitable electrode material for producing functional MEAs.