Publications by authors named "Susanna Narkilahti"

56 Publications

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

Sci Data 2022 03 30;9(1):120. Epub 2022 Mar 30.

Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

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.
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http://dx.doi.org/10.1038/s41597-022-01242-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8969177PMC
March 2022

Human Neurons Form Axon-Mediated Functional Connections with Human Cardiomyocytes in Compartmentalized Microfluidic Chip.

Int J Mol Sci 2022 Mar 15;23(6). Epub 2022 Mar 15.

Neuro Group, Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland.

The cardiac autonomic nervous system (cANS) regulates cardiac function by innervating cardiac tissue with axons, and cardiomyocytes (CMs) and neurons undergo comaturation during the heart innervation in embryogenesis. As cANS is essential for cardiac function, its dysfunctions might be fatal; therefore, cardiac innervation models for studying embryogenesis, cardiac diseases, and drug screening are needed. However, previously reported neuron-cardiomyocyte (CM) coculture chips lack studies of functional neuron-CM interactions with completely human-based cell models. Here, we present a novel completely human cell-based and electrophysiologically functional cardiac innervation on a chip in which a compartmentalized microfluidic device, a 3D3C chip, was used to coculture human induced pluripotent stem cell (hiPSC)-derived neurons and CMs. The 3D3C chip enabled the coculture of both cell types with their respective culture media in their own compartments while allowing the neuronal axons to traverse between the compartments via microtunnels connecting the compartments. Furthermore, the 3D3C chip allowed the use of diverse analysis methods, including immunocytochemistry, RT-qPCR and video microscopy. This system resembled the in vivo axon-mediated neuron-CM interaction. In this study, the evaluation of the CM beating response during chemical stimulation of neurons showed that hiPSC-neurons and hiPSC-CMs formed electrophysiologically functional axon-mediated interactions.
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http://dx.doi.org/10.3390/ijms23063148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8955890PMC
March 2022

A kainic acid-induced seizure model in human pluripotent stem cell-derived cortical neurons for studying the role of IL-6 in the functional activity.

Stem Cell Res 2022 04 17;60:102665. Epub 2022 Jan 17.

Neuro Group, Faculty of Medicine and Health Technology, Tampere University, 33520 Tampere, Finland. Electronic address:

Human pluripotent stem cell (hPSC)-derived neural cultures have attracted interest for modeling epilepsy and seizure-like activity in vitro. Clinical and experimental evidence have shown that the multifunctional inflammatory cytokine interleukin (IL)-6 plays a significant role in epilepsy. However, the role of IL-6 in neuronal networks remains unclear. In this study, we modelled seizure-like activity in hPSC-derived cortical neurons using kainic acid (KA) and explored the effects of IL-6 and its counterpart, hyper-IL-6 (H-IL-6), a fusion protein consisting of IL-6 and its soluble receptor, IL-6R. In the seizure-like model, functionally mature neuronal networks responded to KA induction with an increased bursting phenotype at the single electrode level, while network level bursts decreased. The IL-6 receptors, IL6R and gp130, were expressed in hPSC-derived cortical neurons, and the gene expression of IL6R increased during maturation. Furthermore, the expression of IL-6R increased not only after IL-6 and H-IL-6 treatment but also after KA treatment. Stimulation with IL-6 or H-IL-6 was not toxic to the neurons and cytokine pretreatment did not independently modulate neuronal network activity or KA-induced seizures. Furthermore, the increased expression of IL-6R in response to IL-6, H-IL-6 and KA implies that neurons can respond through both classical and trans-signaling pathways. Acute treatment with IL-6 and H-IL-6 did not alter functional activity, suggesting that IL-6 does not affect the induction or modulation of newly induced seizures in healthy cultures. Overall, we propose this model as a useful tool to study seizure-like activity in neuronal networks in vitro.
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http://dx.doi.org/10.1016/j.scr.2022.102665DOI Listing
April 2022

Bidirectional cell-matrix interaction dictates neuronal network formation in a brain-mimetic 3D scaffold.

Acta Biomater 2022 03 10;140:314-323. Epub 2021 Dec 10.

Bioengineering and Nanomedicine Group, Faculty of Medicine and Health Technology, Tampere University, 33720 Tampere, Finland. Electronic address:

Human pluripotent stem cells (hPSC) derived neurons are emerging as a powerful tool for studying neurobiology, disease pathology, and modeling. Due to the lack of platforms available for housing and growing hPSC-derived neurons, a pressing need exists to tailor a brain-mimetic 3D scaffold that recapitulates tissue composition and favourably regulates neuronal network formation. Despite the progress in engineering biomimetic scaffolds, an ideal brain-mimetic scaffold is still elusive. We bioengineered a physiologically relevant 3D scaffold by integrating brain-like extracellular matrix (ECM) components and chemical cues. Culturing hPSCs-neurons in hyaluronic acid (HA) gels and HA-chondroitin sulfate (HA-CS) composite gels showed that the CS component prevails as the predominant factor for the growth of neuronal cells, albeit to modest efficacy. Covalent grafting of dopamine (DA) moieties to the HA-CS gel (HADA-CS) enhanced the scaffold stability and stimulated the gel's remodeling properties by entrapping cell-secreted laminin, and binding brain-derived neurotrophic factor (BDNF). Neurons cultured in the scaffold expressed Col1, Col11, and ITGB4; important for cell adhesion and cell-ECM signaling. Thus, the HA-CS scaffold with integrated chemical cues (DA) supported neuronal growth and network formation. This scaffold offers a valuable tool for tissue engineering and disease modeling and helps in bridging the gap between animal models and human diseases by providing biomimetic neurophysiology. STATEMENT OF SIGNIFICANCE: Developing a brain mimetic 3D scaffold that supports neuronal growth could potentially be useful to study neurobiology, disease pathology, and disease modeling. However, culturing human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (ESCs) derived neurons in a 3D matrix is extremely challenging as neurons are very sensitive cells and require tailored composition, viscoelasticity, and chemical cues. This article identified the key chemical cues necessary for designing neuronal matrix that trap the cell-produced ECM and neurotrophic factors and remodel the matrix and supports neurite outgrowth. The tailored injectable scaffold possesses self-healing/shear-thinning property which is useful to design injectable gels for regenerative medicine and disease modeling that provides biomimetic neurophysiology.
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http://dx.doi.org/10.1016/j.actbio.2021.12.010DOI Listing
March 2022

Novel method to produce a layered 3D scaffold for human pluripotent stem cell-derived neuronal cells.

J Neurosci Methods 2021 02 17;350:109043. Epub 2020 Dec 17.

NeuroGroup, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

Background: Three-dimensional (3D) in vitro models have been developed into more in vivo resembling structures. In particular, there is a need for human-based models for neuronal tissue engineering (TE). To produce such a model with organized microenvironment for cells in central nervous system (CNS), a 3D layered scaffold composed of hydrogel and cell guiding fibers has been proposed.

New Method: Here, we describe a novel method for producing a layered 3D scaffold consisting of electrospun poly (L,D-lactide) fibers embedded into collagen 1 hydrogel to achieve better resemblance of cells' natural microenvironment for human pluripotent stem cell (hPSC)-derived neurons. The scaffold was constructed via a single layer-by-layer process using an electrospinning technique with a unique collector design.

Results: The method enabled the production of layered 3D cell-containing scaffold in a single process. HPSC-derived neurons were found in all layers of the scaffold and exhibited a typical neuronal phenotype. The guiding fiber layers supported the directed cell growth and extension of the neurites inside the scaffold without additional functionalization.

Comparison With Existing Methods: Previous methods have required several process steps to construct 3D layer-by-layer scaffolds.

Conclusions: We introduced a method to produce layered 3D scaffolds to mimic the cell guiding cues in CNS by alternating the soft hydrogel matrix and fibrous guidance cues. The produced scaffold successfully enabled the long-term culture of hPSC-derived neuronal cells. This layered 3D scaffold is a useful model for in vitro and in vivo neuronal TE applications.
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http://dx.doi.org/10.1016/j.jneumeth.2020.109043DOI Listing
February 2021

Oxygen-Glucose Deprivation-Induced Stroke Models with Human Neuroblastoma Cell- and Induced Pluripotent Stem Cell-Derived Neurons.

Stem Cells Int 2020 29;2020:8841026. Epub 2020 Oct 29.

Adult Stem Cell Group, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

Stroke is a devastating neurological disorder and one of the leading causes of mortality and disability. To understand the cellular and molecular mechanisms of stroke and to develop novel therapeutic approaches, two different human cell-based stroke models were established using oxygen-glucose deprivation (OGD) conditions. In addition, the effect of adipose stem cells (ASCs) on OGD-induced injury was studied. In the present study, SH-SY5Y human neuroblastoma cells and human induced pluripotent stem cells (hiPSCs) were differentiated into neurons, cultured under OGD conditions (1% O) for 24 h, and subjected to a reperfusion period for 24 or 72 h. After OGD, ASCs were cocultured with neurons on inserts for 24 or 72 h to study the neuroprotective potential of ASCs. The effect of OGD and ASC coculture on the viability, apoptosis, and proliferation of and axonal damage to neuronal cells was studied. The results showed that OGD conditions induced cytotoxicity and apoptosis of SH-SY5Y- and hiPSC-derived neurons, although more severe damage was detected in SH-SY5Y-derived neurons than in hiPSC-derived neurons. Coculture with ASCs was protective for neurons, as the number of dead ASC-cocultured neurons was lower than that of control cells, and coculture increased the proliferation of both cell types. To conclude, we developed human cell-based stroke models in SH-SY5Y- and hiPSC-derived neurons. This was the first time hiPSCs were used to model stroke . Since OGD had different effects on the studied cell types, this study highlights the importance of using several cell types in studies to confirm the outcomes of the study. Here, ASCs exerted a neuroprotective effect by increasing the proliferation and decreasing the death of SH-SY5Y- and hiPSC-derived neurons after OGD.
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http://dx.doi.org/10.1155/2020/8841026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647751PMC
October 2020

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

Biosens Bioelectron 2020 Nov 26;168:112553. Epub 2020 Aug 26.

NeuroGroup, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bios.2020.112553DOI Listing
November 2020

Covalent immobilization of luminescent oxygen indicators reduces cytotoxicity.

Biomed Microdevices 2020 06 3;22(2):41. Epub 2020 Jun 3.

Faculty of Medicine and Health Technology, Tampere University, Korkeakoulunkatu 3, 33720, Tampere, Finland.

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.
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http://dx.doi.org/10.1007/s10544-020-00495-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270993PMC
June 2020

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

Micromachines (Basel) 2020 May 14;11(5). Epub 2020 May 14.

Micro and Nanosystems Research Group, Faculty of Medicine and Health Technology, Tampere University, 33720 Tampere, Finland.

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.
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http://dx.doi.org/10.3390/mi11050497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281740PMC
May 2020

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

Sci Rep 2019 11 20;9(1):17125. Epub 2019 Nov 20.

Faculty of Medicine and Health Technology and BioMediTech, Tampere University, Tampere, Finland.

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.
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http://dx.doi.org/10.1038/s41598-019-53647-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868015PMC
November 2019

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

Sci Rep 2019 11 15;9(1):16944. Epub 2019 Nov 15.

NeuroGroup, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

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.
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http://dx.doi.org/10.1038/s41598-019-53414-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858358PMC
November 2019

Screening of Hydrogels for Human Pluripotent Stem Cell-Derived Neural Cells: Hyaluronan-Polyvinyl Alcohol-Collagen-Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold.

Macromol Biosci 2019 07 7;19(7):e1900096. Epub 2019 Jun 7.

NeuroGroup, Faculty of Medicine and Health Technology, Tampere University, Kalevantie 4, 33014, Tampere, Finland.

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell-, biomaterial-, and environment-related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan-poly(vinyl alcohol) (HA1-PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1-PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1-PVA-col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D.
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http://dx.doi.org/10.1002/mabi.201900096DOI Listing
July 2019

Advances in Human Stem Cell-Derived Neuronal Cell Culturing and Analysis.

Adv Neurobiol 2019 ;22:299-329

NeuroGroup, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

This chapter provides an overview of the current stage of human in vitro functional neuronal cultures, their biological application areas, and modalities to analyze their behavior. During the last 10 years, this research area has changed from being practically non-existent to one that is facing high expectations. Here, we present a case study as a comprehensive short history of this process based on extensive studies conducted at NeuroGroup (University of Tampere) and Computational Biophysics and Imaging Group (Tampere University of Technology), ranging from the differentiation and culturing of human pluripotent stem cell (hPSC)-derived neuronal networks to their electrophysiological analysis. After an introduction to neuronal differentiation in hPSCs, we review our work on their functionality and approaches for extending cultures from 2D to 3D systems. Thereafter, we discuss our target applications in neuronal developmental modeling, toxicology, drug screening, and disease modeling. The development of signal analysis methods was required due to the unique functional and developmental properties of hPSC-derived neuronal cells and networks, which separate them from their much-used rodent counterparts. Accordingly, a line of microelectrode array (MEA) signal analysis methods was developed. This work included the development of action potential spike detection methods, entropy-based methods and additional methods for burst detection and quantification, joint analysis of spikes and bursts to analyze the spike waveform compositions of bursts, assessment methods for network synchronization, and computational simulations of synapses and neuronal networks.
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http://dx.doi.org/10.1007/978-3-030-11135-9_13DOI Listing
August 2019

Catalytically inactive carbonic anhydrase-related proteins enhance transport of lactate by MCT1.

FEBS Open Bio 2019 07 11;9(7):1204-1211. Epub 2019 Jun 11.

Division of General Zoology, FB Biologie, TU Kaiserslautern, Germany.

Carbonic anhydrases (CA) catalyze the reversible hydration of CO to protons and bicarbonate and thereby play a fundamental role in the epithelial acid/base transport mechanisms serving fluid secretion and absorption for whole-body acid/base regulation. The three carbonic anhydrase-related proteins (CARPs) VIII, X, and XI, however, are catalytically inactive. Previous work has shown that some CA isoforms noncatalytically enhance lactate transport through various monocarboxylate transporters (MCT). Therefore, we examined whether the catalytically inactive CARPs play a role in lactate transport. Here, we report that CARP VIII, X, and XI enhance transport activity of the MCT MCT1 when coexpressed in Xenopus oocytes, as evidenced by the rate of rise in intracellular H+ concentration detected using ion-sensitive microelectrodes. Based on previous studies, we suggest that CARPs may function as a 'proton antenna' for MCT1, to drive proton-coupled lactate transport across the cell membrane.
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http://dx.doi.org/10.1002/2211-5463.12647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609565PMC
July 2019

Microelectrode Array With Transparent ALD TiN Electrodes.

Front Neurosci 2019 22;13:226. Epub 2019 Mar 22.

Micro- and Nanosystems Research Group, BioMediTech Institute and Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

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.
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http://dx.doi.org/10.3389/fnins.2019.00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438859PMC
March 2019

Ion Beam Assisted E-Beam Deposited TiN Microelectrodes-Applied to Neuronal Cell Culture Medium Evaluation.

Front Neurosci 2018 4;12:882. Epub 2018 Dec 4.

BioMediTech Institute and Faculty of Biomedical Sciences and Engineering, Tampere University of Technology, Tampere, Finland.

Microelectrode material and cell culture medium have significant roles in the signal-to-noise ratio and cell well-being in electrophysiological studies. Here, we report an ion beam assisted e-beam deposition (IBAD) based process as an alternative titanium nitride (TiN) deposition method for sputtering in the fabrication of state-of-the-art TiN microelectrode arrays (MEAs). The effects of evaporation and nitrogen flow rates were evaluated while developing the IBAD TiN deposition process. Moreover, the produced IBAD TiN microelectrodes were characterized by impedance, charge transfer capacity (CTC) and noise measurements for electrical properties, AFM and SEM for topological imaging, and EDS for material composition. The impedance (at 1 kHz) of brand new 30 μm IBAD TiN microelectrodes was found to be double but still below 100 kΩ compared with commercial reference MEAs with sputtered TiN microelectrodes of the same size. On the contrary, the noise level of IBAD TiN MEAs was lower compared with that of commercial sputtered TiN MEAs in equal conditions. In CTC IBAD TiN electrodes (3.3 mC/cm) also outperformed the sputtered counterparts (2.0 mC/cm). To verify the suitability of IBAD TiN microelectrodes for cell measurements, human pluripotent stem cell (hPSC)-derived neuronal networks were cultured on IBAD TiN MEAs and commercial sputtered TiN MEAs in two different media: neural differentiation medium (NDM) and BrainPhys (BPH). The effect of cell culture media to hPSC derived neuronal networks was evaluated to gain more stable and more active networks. Higher spontaneous activity levels were measured from the neuronal networks cultured in BPH compared with those in NDM in both MEA types. However, BPH caused more problems in cell survival in long-term cultures by inducing neuronal network retraction and clump formation after 1-2 weeks. In addition, BPH was found to corrode the SiN insulator layer more than NDM medium. The developed IBAD TiN process gives MEA manufacturers more choices to choose which method to use to deposit TiN electrodes and the medium evaluation results remind that not only electrode material but also insulator layer and cell culturing medium have crucial role in successful long term MEA measurements.
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http://dx.doi.org/10.3389/fnins.2018.00882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6290344PMC
December 2018

Effects of inflammatory cytokines IFN-γ, TNF-α and IL-6 on the viability and functionality of human pluripotent stem cell-derived neural cells.

J Neuroimmunol 2019 06 29;331:36-45. Epub 2018 Aug 29.

Neuro Group, BioMeditech, The Faculty of Medicine and Life Sciences, University of Tampere, Finland. Electronic address:

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease, where neural progenitor cell (NPC) transplantation has been suggested as a potential neuroprotective therapeutic strategy. Since the effect of inflammation on NPCs is poorly known, their effect on the survival and functionality of human NPCs were studied. Treatment with interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ did not induced cytotoxicity, but IFN-γ treatment showed decreased proliferation and neuronal migration. By contrast, increased proliferation and inhibition of electrical activity were detected after TNF-α treatment. Treatments induced secretion of inflammatory factors. Inflammatory cytokines appear to modulate proliferation as well as the cellular and functional properties of human NPCs.
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http://dx.doi.org/10.1016/j.jneuroim.2018.07.010DOI Listing
June 2019

GABA and Gap Junctions in the Development of Synchronized Activity in Human Pluripotent Stem Cell-Derived Neural Networks.

Front Cell Neurosci 2018 6;12:56. Epub 2018 Mar 6.

NeuroGroup Laboratory, BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.

The electrical activity of the brain arises from single neurons communicating with each other. However, how single neurons interact during early development to give rise to neural network activity remains poorly understood. We studied the emergence of synchronous neural activity in human pluripotent stem cell (hPSC)-derived neural networks simultaneously on a single-neuron level and network level. The contribution of gamma-aminobutyric acid (GABA) and gap junctions to the development of synchronous activity in hPSC-derived neural networks was studied with GABA agonist and antagonist and by blocking gap junctional communication, respectively. We characterized the dynamics of the network-wide synchrony in hPSC-derived neural networks with high spatial resolution (calcium imaging) and temporal resolution microelectrode array (MEA). We found that the emergence of synchrony correlates with a decrease in very strong GABA excitation. However, the synchronous network was found to consist of a heterogeneous mixture of synchronously active cells with variable responses to GABA, GABA agonists and gap junction blockers. Furthermore, we show how single-cell distributions give rise to the network effect of GABA, GABA agonists and gap junction blockers. Finally, based on our observations, we suggest that the earliest form of synchronous neuronal activity depends on gap junctions and a decrease in GABA induced depolarization but not on GABA mediated signaling.
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http://dx.doi.org/10.3389/fncel.2018.00056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845705PMC
March 2018

Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures.

Stem Cell Res 2018 03 31;27:151-161. Epub 2018 Jan 31.

NeuroGroup, BioMediTech and Faculty of Medicine and Life Sciences, University of Tampere, Arvo Ylpön katu 34, 33520 Tampere, Finland. Electronic address:

Long-term neural differentiation of human pluripotent stem cells (hPSCs) is associated with enhanced neuronal maturation, which is a necessity for creation of representative in vitro models. It also induces neurogenic-to-gliogenic fate switch, increasing proportion of endogenous astrocytes formed from the common neural progenitors. However, the significance of prolonged differentiation on the neural cell type composition and functional development of hPSC-derived neuronal cells has not been well characterized. Here, we studied two hPSC lines, both of which initially showed good neuronal differentiation capacity. However, the propensity for endogenous astrogenesis and maturation state after extended differentiation varied. Live cell calcium imaging revealed that prolonged differentiation facilitated maturation of GABAergic signaling. According to extracellular recordings with microelectrode array (MEA), neuronal activity was limited to fewer areas of the culture, which expressed more frequent burst activity. Efficient maturation after prolonged differentiation also promoted organization of spontaneous activity by burst compaction. These results suggest that although prolonged neural differentiation can be challenging, it has beneficial effect on functional maturation, which can also improve transition to different neural in vitro models and applications.
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http://dx.doi.org/10.1016/j.scr.2018.01.018DOI Listing
March 2018

Optimised PDMS Tunnel Devices on MEAs Increase the Probability of Detecting Electrical Activity from Human Stem Cell-Derived Neuronal Networks.

Front Neurosci 2017 31;11:606. Epub 2017 Oct 31.

NeuroGroup, BioMediTech Institute and Faculty of Medicine and Biosciences, University of Tampere, Tampere, Finland.

Measurement of the activity of human pluripotent stem cell (hPSC)-derived neuronal networks with microelectrode arrays (MEAs) plays an important role in functional brain modelling and in neurotoxicological screening. The previously reported hPSC-derived neuronal networks do not, however, exhibit repeatable, stable functional network characteristics similar to rodent cortical cultures, making the interpretation of results difficult. In earlier studies, microtunnels have been used both to control and guide cell growth and amplify the axonal signals of rodent neurons. The aim of the current study was to develop tunnel devices that would facilitate signalling and/or signal detection in entire hPSC-derived neuronal networks containing not only axons, but also somata and dendrites. Therefore, MEA-compatible polydimethylsiloxane (PDMS) tunnel devices with 8 different dimensions were created. The hPSC-derived neurons were cultured in the tunnel devices on MEAs, and the spontaneous electrical activity of the networks was measured for 5 weeks. Although the tunnel devices improved the signal-to-noise ratio only by 1.3-fold at best, they significantly increased the percentage of electrodes detecting neuronal activity (52-100%) compared with the controls (27%). Significantly higher spike and burst counts were also obtained using the tunnel devices. Neuronal networks inside the tunnels were amenable to pharmacological manipulation. The results suggest that tunnel devices encompassing the entire neuronal network can increase the measured spontaneous activity in hPSC-derived neuronal networks on MEAs. Therefore, they can increase the efficiency of functional studies of hPSC-derived networks on MEAs.
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http://dx.doi.org/10.3389/fnins.2017.00606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671636PMC
October 2017

Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro.

Stem Cell Res 2017 10 12;24:118-127. Epub 2017 Sep 12.

NeuroGroup, BioMediTech, University of Tampere, Lääkärinkatu 1, 33520 Tampere, Finland.

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511-E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also for the production of clinical grade hPSC-derived cells for regenerative medicine applications.
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http://dx.doi.org/10.1016/j.scr.2017.09.002DOI Listing
October 2017

Direct Laser Writing of Tubular Microtowers for 3D Culture of Human Pluripotent Stem Cell-Derived Neuronal Cells.

ACS Appl Mater Interfaces 2017 Aug 31;9(31):25717-25730. Epub 2017 Jul 31.

Biomaterials and Tissue Engineering Group, BioMediTech and Faculty of Biomedical Sciences and Engineering, Tampere University of Technology , Korkeakoulunkatu 3, 33720 Tampere, Finland.

As the complex structure of nervous tissue cannot be mimicked in two-dimensional (2D) cultures, the development of three-dimensional (3D) neuronal cell culture platforms is a topical issue in the field of neuroscience and neural tissue engineering. Computer-assisted laser-based fabrication techniques such as direct laser writing by two-photon polymerization (2PP-DLW) offer a versatile tool to fabricate 3D cell culture platforms with highly ordered geometries in the size scale of natural 3D cell environments. In this study, we present the design and 2PP-DLW fabrication process of a novel 3D neuronal cell culture platform based on tubular microtowers. The platform facilitates efficient long-term 3D culturing of human neuronal cells and supports neurite orientation and 3D network formation. Microtower designs both with or without intraluminal guidance cues and/or openings in the tower wall are designed and successfully fabricated from Ormocomp. Three of the microtower designs are chosen for the final culture platform: a design with openings in the wall and intralumial guidance cues (webs and pillars), a design with openings but without intraluminal structures, and a plain cylinder design. The proposed culture platform offers a promising concept for future 3D cultures in the field of neuroscience.
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http://dx.doi.org/10.1021/acsami.7b05536DOI Listing
August 2017

Aligned Poly(ε-caprolactone) Nanofibers Guide the Orientation and Migration of Human Pluripotent Stem Cell-Derived Neurons, Astrocytes, and Oligodendrocyte Precursor Cells In Vitro.

Macromol Biosci 2017 07 15;17(7). Epub 2017 Mar 15.

NeuroGroup, BioMediTech and Faculty of Medicine and Life Sciences, University of Tampere, Lääkärinkatu 1, FI-33520, Tampere, Finland.

Stem cell transplantations for spinal cord injury (SCI) have been studied extensively for the past decade in order to replace the damaged tissue with human pluripotent stem cell (hPSC)-derived neural cells. Transplanted cells may, however, benefit from supporting and guiding structures or scaffolds in order to remain viable and integrate into the host tissue. Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. All cell types are shown to efficiently spread over the nanofiber platform and orient according to the fiber alignment. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment. In this work, clinically relevant materials and substrates for nanofibers, fiber coatings, and hydrogel scaffolds are used and combined with cells suitable for developing functional cell grafts for SCI repair.
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http://dx.doi.org/10.1002/mabi.201600517DOI Listing
July 2017

Bioamine-crosslinked gellan gum hydrogel for neural tissue engineering.

Biomed Mater 2017 03 24;12(2):025014. Epub 2017 Mar 24.

BioMediTech Institute and Faculty of Biomedical Sciences and Engineering, Tampere University of Technology, Korkeakoulunkatu 3, FI-33720 Tampere, Finland. BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Lääkärinkatu 1, FI-33520 Tampere, Finland.

Neural tissue engineering and three-dimensional in vitro tissue modeling require the development of biomaterials that take into account the specified requirements of human neural cells and tissue. In this study, an alternative method of producing biomimetic hydrogels based on gellan gum (GG) was developed by replacing traditional crosslinking methods with the bioamines spermidine and spermine. These bioamines were proven to function as crosslinkers for GG hydrogel at +37 °C, allowing for the encapsulation of human neurons. We studied the mechanical and rheological properties of the formed hydrogels, which showed biomimicking properties comparable to naïve rabbit brain tissue under physiologically relevant stress and strain. Human pluripotent stem cell-derived neuronal cells demonstrated good cytocompatibility in the GG-based hydrogels. Moreover, functionalization of GG hydrogels with laminin resulted in cell type-specific behavior: neuronal cell maturation and neurite migration.
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http://dx.doi.org/10.1088/1748-605X/aa62b0DOI Listing
March 2017

Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array.

J Neurosci Methods 2017 03 1;280:27-35. Epub 2017 Feb 1.

Tampere University of Technology, Biomedical Sciences and Engineering, BioMediTech, Korkeakoulunkatu 3, FI-33720, Tampere, Finland. Electronic address:

Background: Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential.

New Method: We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber.

Results: We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments.

Comparison With Existing Methods: Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO gas. Utilizing dry gas supply makes the proposed chamber simple to use.

Conclusion: Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture.
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http://dx.doi.org/10.1016/j.jneumeth.2017.01.019DOI Listing
March 2017

Simulation of developing human neuronal cell networks.

Biomed Eng Online 2016 Aug 30;15(1):105. Epub 2016 Aug 30.

Department of Electronics and Communications Engineering, Tampere University of Technology, BioMediTech, PL100, Tampere, Finland.

Background: Microelectrode array (MEA) is a widely used technique to study for example the functional properties of neuronal networks derived from human embryonic stem cells (hESC-NN). With hESC-NN, we can investigate the earliest developmental stages of neuronal network formation in the human brain.

Methods: In this paper, we propose an in silico model of maturating hESC-NNs based on a phenomenological model called INEX. We focus on simulations of the development of bursts in hESC-NNs, which are the main feature of neuronal activation patterns. The model was developed with data from developing hESC-NN recordings on MEAs which showed increase in the neuronal activity during the investigated six measurement time points in the experimental and simulated data.

Results: Our simulations suggest that the maturation process of hESC-NN, resulting in the formation of bursts, can be explained by the development of synapses. Moreover, spike and burst rate both decreased at the last measurement time point suggesting a pruning of synapses as the weak ones are removed.

Conclusions: To conclude, our model reflects the assumption that the interaction between excitatory and inhibitory neurons during the maturation of a neuronal network and the spontaneous emergence of bursts are due to increased connectivity caused by the forming of new synapses.
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http://dx.doi.org/10.1186/s12938-016-0226-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006268PMC
August 2016

Increased plasma UCH-L1 after aneurysmal subarachnoid hemorrhage is associated with unfavorable neurological outcome.

J Neurol Sci 2016 Feb 29;361:144-9. Epub 2015 Dec 29.

Department of Neurosciences and Rehabilitation, Tampere University Hospital, Tampere, Finland.

Objective: Aneurysmal subarachnoid hemorrhage (aSAH) is a common cause of long-term disability and death. After primary hemorrhage, secondary brain injury is the main cause of mortality and morbidity. Despite extensive research, reliable prognostic biomarkers are lacking. We measured ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) levels in aSAH patients to evaluate its prognostic potential. This is the first time that plasma UCH-L1 has been studied as a potential prognostic biomarker in patients with aSAH.

Methods: In this prospective population-based study, UCH-L1 levels were measured in aSAH patients (n=47) for up to five days. UCH-L1 was measured at 0, 12 and 24h after the admission to the intensive care unit (ICU) and daily thereafter until the patient was transferred from the ICU. Only patients whose UCH-L1 was measured within 24h from aSAH were included in the study. The patients' neurological outcome was evaluated with the modified Rankin Scale (mRS) at six months after aSAH.

Results: UCH-L1 levels during the first 24h after aSAH were not significantly different between the groups with favorable (mRS 0-2) and unfavorable (mRS 3-6) neurological outcome. In 22 patients, UCH-L1 levels were obtained for up to five days. In this subgroup, UCH-L1 measured at day five showed significant elevation from baseline levels in patients with unfavorable outcome (p=0.026). Elevated UCH-L1 levels at day five were higher in patients with unfavorable outcome than in patients with favorable outcome (p=0.001).

Conclusions: Elevated UCH-L1 levels during the five-day follow-up were associated with unfavorable neurological outcome. Repetitive measurements of UCH-L1 concentrations with an emphasis on change relative to the individual baseline could be the optimal approach for future clinical studies.
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http://dx.doi.org/10.1016/j.jns.2015.12.046DOI Listing
February 2016

Joint analysis of extracellular spike waveforms and neuronal network bursts.

J Neurosci Methods 2016 Feb 7;259:143-155. Epub 2015 Dec 7.

Tampere University of Technology, Department of Electronics and Communications Engineering, Computational Biophysics and Imaging Group, BioMediTech, Biokatu 6, FI-33520 Tampere, Finland. Electronic address:

Background: Neuronal networks are routinely assessed based on extracellular electrophysiological microelectrode array (MEA) measurements by spike sorting, and spike and burst statistics. We propose to jointly analyze sorted spikes and detected bursts, and hypothesize that the obtained spike type compositions of the bursts can provide new information on the functional networks.

New Method: Spikes are detected and sorted to obtain spike types and bursts are detected. In the proposed joint analysis, each burst spike is associated with a spike type, and the spike type compositions of the bursts are assessed.

Results: The proposed method was tested with simulations and MEA measurements of in vitro human stem cell derived neuronal networks under different pharmacological treatments. The results show that the treatments altered the spike type compositions of the bursts. For example, 6-cyano-7-nitroquinoxaline-2,3-dione almost completely abolished two types of spikes which had composed the bursts in the baseline, while bursts of spikes of two other types appeared more frequently. This phenomenon was not observable by spike sorting or burst analysis alone, but was revealed by the proposed joint analysis.

Comparison With Existing Methods: The existing methods do not provide the information obtainable with the proposed method: for the first time, the spike type compositions of bursts are analyzed.

Conclusions: We showed that the proposed method provides useful and novel information, including the possible changes in the spike type compositions of the bursts due to external factors. Our method can be employed on any data exhibiting sortable action potential waveforms and detectable bursts.
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http://dx.doi.org/10.1016/j.jneumeth.2015.11.022DOI Listing
February 2016

Healthy human CSF promotes glial differentiation of hESC-derived neural cells while retaining spontaneous activity in existing neuronal networks.

Biol Open 2013 Jun 13;2(6):605-12. Epub 2013 May 13.

Critical Care Medicine Research Group, Department of Intensive Care Unit, Tampere University Hospital , FI-33521 Tampere , Finland ; NeuroGroup, Institute of Biomedical Technology/BioMediTech, University of Tampere , FI-33520 Tampere , Finland.

The possibilities of human pluripotent stem cell-derived neural cells from the basic research tool to a treatment option in regenerative medicine have been well recognized. These cells also offer an interesting tool for in vitro models of neuronal networks to be used for drug screening and neurotoxicological studies and for patient/disease specific in vitro models. Here, as aiming to develop a reductionistic in vitro human neuronal network model, we tested whether human embryonic stem cell (hESC)-derived neural cells could be cultured in human cerebrospinal fluid (CSF) in order to better mimic the in vivo conditions. Our results showed that CSF altered the differentiation of hESC-derived neural cells towards glial cells at the expense of neuronal differentiation. The proliferation rate was reduced in CSF cultures. However, even though the use of CSF as the culture medium altered the glial vs. neuronal differentiation rate, the pre-existing spontaneous activity of the neuronal networks persisted throughout the study. These results suggest that it is possible to develop fully human cell and culture-based environments that can further be modified for various in vitro modeling purposes.
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http://dx.doi.org/10.1242/bio.20134648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683163PMC
June 2013

Comparative analysis of targeted differentiation of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells reveals variability associated with incomplete transgene silencing in retrovirally derived hiPSC lines.

Stem Cells Transl Med 2013 Feb 22;2(2):83-93. Epub 2013 Jan 22.

Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Helsinki, Finland.

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.
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http://dx.doi.org/10.5966/sctm.2012-0047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659749PMC
February 2013
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