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.

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-1β (IL-1β) 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.

No abstract

The polarized morphology of neurons allows the transmission of neuronal signals along long, slender axons over extended distances. The dysfunction and degeneration of axons are important hallmarks of many neurological disorders and traumas ranging from spinal cord injury to neurodegenerative diseases such as Alzheimer's disease. Thus, targeted research on axons is of great importance for improving the understanding of central nervous system (CNS) diseases and developing treatments for these devastating conditions, many of which lack disease-alleviating or disease-preventing therapies. [1,2] Human pluripotent stem cell (hPSC)-derived neural cells hold great promise for in vitro disease modeling and drug discovery for CNS diseases. [3,4] hPSCs provide an unlimited cell source for producing several types of neurons, and induced pluripotent stem cell (iPSC) technology enables the generation of patient-derived neurons that can recapitulate disease characteristics in vitro. [5,6] hPSC-based models have been used to study CNS diseases associated with axonal dysfunction and degeneration. [7][8][9] However, the full potential of in vitro modeling requires combining hPSC biology with state-of-the-art engineering technologies.Axonal research has been remarkably accelerated by the development of engineered in vitro devices that guide the organization of neurons, allowing the isolation of the axonal microenvironment. These compartmentalized devices enable precise spatial control, for example, targeted monitoring, measurement, and manipulation of axons, which are unfeasible or difficult to perform with conventional in vitro culture systems or in vivo. [10][11][12] The first devices used for neuron compartmentalization were Campenot chambers, which use a Teflon ring for the separation of neuronal somas and axons. [13][14][15] These were followed by microfluidic polydimethylsiloxane (PDMS)based devices, which currently represent the most common device type owing to their ease of fabrication and possibility of producing complex and highly controllable devices. [10,11,[16][17][18][19] Axonal isolation in PDMS microfluidic devices is based on microtunnels whose dimensions allow the passage of axons Axonal dysfunction and degeneration are important pathological features of central nervous system (CNS) diseases and traumas, such as Alzheimer's disease, traumatic brain injury, ischemic stroke and spinal cord injury. Engineered microfluidic chips combined with human pluripotent stem cell (hPSC)-derived neurons provide valuable tools for targeted in vitro research on axons to improve understanding of disease mechanisms and enhance drug development. Here, a polydimethylsiloxane (PDMS) microfluidic chip integrated with a light patterned substrate is utilized to achieve both isolated and unidirectional axonal growth of hPSC-derived neurons. The isolation of axons from somas and dendrites and robust axonal outgrowth to adjacent, axonal compartment, is achieved by optimized cross-sectional area and length of PDMS microtunnels in the microflu...

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.