Human Pluripotent Stem Cell-Derived Neurons to Model Epilepsy

Epilepsia on monimuotoinen neurologinen sairaus, johon liittyy toistuvia ennalta arvaamattomia kohtauksia. Epileptiset kohtaukset syntyvät aivojen hermosolujen sähköisen toiminnan häiriöistä. Sähköisten häiriöiden syy on suurelta osin tuntematon, mutta erilaiset ärsykkeet, kuten rakenteelliset, metaboliset, immunologiset tai geneettiset muutokset, voivat johtaa epilepsian puhkeamiseen. Maailmassa yli 70 miljoonaa ihmistä sairastaa epilepsiaa ja sairautta esiintyy kaikissa ikäryhmissä. Epilepsiaan ei ole toistaiseksi olemassa parannuskeinoa. Vaikka suurin osa potilaista saa lievitystä oireisiin kohtauksia ehkäisevällä lääkityksellä, noin 30 % potilaista ei hyödy lääkityksestä. Suurin osa lääketutkimuksista tehdään erilaisilla eläinmalleilla, mutta niiden soveltaminen ihmisiin on osoittautunut haastavaksi. Sen vuoksi on välttämätöntä kehittää ihmisperäisiä prekliinisiä epilepsiamalleja. Tämä väitöskirjatutkimus keskittyi kehittämään ihmisperäisiä kohtausmalleja maljalla hyödyntäen ihmisen erittäin monikykyisistä kantasoluista johdettuja hermoverkkoja. Kehitetyissä malleissa tutkittiin epilepsian keskeisiä patologisia mekanismeja, joihin kuuluivat epilepsiaan liittyvät geneettiset ja tulehdukselliset tekijät sekä ihmisaivojen yhteyksien jäljittely. Tätä varten erittäin monikykyiset kantasolut erilaistettiin epilepsian kannalta keskeisiksi hermosolutyypeiksi, jotka ovat eksitatoriset (glutamaatti välittäjäainetta tuottavat) ja inhibitoriset (GABA-välittäjäainetta tuottavat) hermosolut. Aivojen hermoverkkojen keskinäisen kytkeytyneisyyden jäljittelemiseksi kehitettiin aivokudossirumalli, jossa voi tutkia hermoverkkojen yhteyksiä ja sähköisten kohtausten kaltaisia tapahtumia. Kohtauksen kaltaiset tapahtumat aiheutettiin hermosoluille kemiallisesti kainaatilla. Tässä tutkimuksessa kohtauksen kaltaisia tapahtumia tutkittiin verkostotasolla. Lisäksi kehitetyssä mallissa arvioitiin keskeisesti epileptisiin kohtauksiin liitetyn tulehduksellisen tekijän interleukiini-6 (IL-6) vaikutusta. Lopuksi keskityttiin epilepsian geneettiseen muodon, Dravetin oireyhtymän (DS), mallintamiseen, jotta voitaisiin arvioida genotyypin ja fenotyypin välistä yhteyttä ja vaikutusta hermosolujen toiminnallisuuteen. Tutkimuksen tulokset osoittivat, että kantasoluista johdetuissa hermoverkoissa voidaan havaita epileptisen kohtauksen kaltaista sähköistä häiriötä kainaattikäsittelyn seurauksena. Tämä viittaa siihen, että mallia voidaan käyttää perustana sairauden muiden näkökohtien tutkimuksessa. Kehitetyn aivokudossirumallin osoitettiin tukevan hermosolujen rakenteellista ja toiminnallista kehitystä. Kainaattikäsittelyn seurauksena aivokudossirun hermoverkot jäljittelivät paikallista kohtaustyyppiä. Hermoverkkojen käsittely kainaatilla ja IL-6:lla nosti IL-6-spesifisen reseptorin ilmentymistä, mutta toiminnallisella tasolla ei havaittu muutoksia hermosolujen aktiivisuudessa. Lopuksi DS-potilaista johdettuja kantasoluja erilaistettiin inhibitorisiksi hermosoluiksi tai hermosolujen sekaviljelmiksi, jolloin pystyttiin osoittamaan potilaskohtaisia eroja toiminnallisuudessa. Tämä paljasti kohtauksen kaltaisia tapahtumia, jotka olivat rinnastettavissa potilaiden kliiniseen tilaan. Johtopäätöksenä voidaan todeta, että tässä väitöskirjatyössä kuvataan merkittävä ihmisperäinen epilepsian solumalli, joka on lupaava työkalu tulevaisuuden epilepsiatutkimuksiin.Epilepsy is a complex neurological disorder that is associated with unpredictable reoccurring seizures. The seizures are triggered by abnormal neuronal activity within the brain. The cause of the seizures is largely unknown, however, a range of insults, such as structural, metabolic, immunological or genetic alterations can lead to the onset of seizures. The disorder is known to affect over 70 million people of all ages globally. To date there is no cure for epilepsy. Though the majority of the patients obtain symptom relief from the anti-seizure medication, about 30% of patients do not benefit from them. Most of the pharmaceutical research is conducted in various animal models, however the translatability to humans has proven challenging. Therefore, the need for human predictive pre-clinical models stands as a necessity. The focus of this thesis was to establish relevant and human-specific seizure models in vitro by using human pluripotent stem cell (hPSC)-derived neuronal networks. In the developed models, key pathological aspects were studied including genetic background and inflammation associated with epilepsy, and mimicry of human brain connectivity. For this reason, hPSCs were firstly differentiated into the appropriate neuronal cell-types which are excitatory- (glutamate producing) and inhibitory (GABA producing) neurons known to be affected in epilepsy. To mimic inter-connectivity of the brain, a microphysiological platform, presented as brain-on-a-chip, was developed to create physiologically relevant interconnected networks and study seizure like events. The seizure-like events were induced chemically on hPSC-derived neurons using kainic acid (KA). Here, seizure-like events were captured at network level. Furthermore, the role of key inflammatory mediator interleukin-6 (IL-6) cytokine, which known to have an active role following onset of seizures, was assessed in the developed model. Finally, focus was set on a genetic form of epilepsy, Dravet syndrome (DS), to assess and characterize the genotype-to-phenotype correlation at a functional level. The overall results presented in this thesis, showed that hPSC-derived neuronal networks display a prominent seizure-like phenotype following response to the seizure inducing agent, KA. This suggests the efficacy of the model as a foundation to investigate further aspects of the disease. The established microphysiological platform incorporated with the disease relevant-cell types, demonstrated support of neuronal structural and functional development. Upon KA induction, hPSC-derived neuronal networks in the platform mimicked a more focal seizure-type. Assessment of IL-6 on KA induced cultures, showed that IL-6 specific receptor, IL-6R, was increased, however at the functional level, changes in activity behavior were not detected. Finally, DS patient-derived hiPSC-neurons when cultured as either GABAergic or mixed neurons, displayed patient specific differences in functional behavior which unveiled distinct seizure-like phenotypes that corresponded to their clinical state. In conclusion, the work of this thesis describes a significant human-based platform that holds promise in advancing future studies related to epilepsy research

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

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.acceptedVersionPeer reviewe

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 ITOto 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 O30 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 O30 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.publishedVersionPeer reviewe

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

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.publishedVersionPeer reviewe

Astrocytes have the capacity to act as antigen-presenting cells in the Parkinson's disease brain

Background Many lines of evidence suggest that accumulation of aggregated alpha-synuclein (αSYN) in the Parkinson’s disease (PD) brain causes infiltration of T cells. However, in which ways the stationary brain cells interact with the T cells remain elusive. Here, we identify astrocytes as potential antigen-presenting cells capable of activating T cells in the PD brain. Astrocytes are a major component of the nervous system, and accumulating data indicate that astrocytes can play a central role during PD progression. Methods To investigate the role of astrocytes in antigen presentation and T-cell activation in the PD brain, we analyzed post mortem brain tissue from PD patients and controls. Moreover, we studied the capacity of cultured human astrocytes and adult human microglia to act as professional antigen-presenting cells following exposure to preformed αSYN fibrils. Results Our analysis of post mortem brain tissue demonstrated that PD patients express high levels of MHC-II, which correlated with the load of pathological, phosphorylated αSYN. Interestingly, a very high proportion of the MHC-II co-localized with astrocytic markers. Importantly, we found both perivascular and infiltrated CD4+ T cells to be surrounded by MHC-II expressing astrocytes, confirming an astrocyte T cell cross-talk in the PD brain. Moreover, we showed that αSYN accumulation in cultured human astrocytes triggered surface expression of co-stimulatory molecules critical for T-cell activation, while cultured human microglia displayed very poor antigen presentation capacity. Notably, intercellular transfer of αSYN/MHC-II deposits occurred between astrocytes via tunneling nanotubes, indicating spreading of inflammation in addition to toxic protein aggregates. Conclusions In conclusion, our data from histology and cell culture studies suggest an important role for astrocytes in antigen presentation and T-cell activation in the PD brain, highlighting astrocytes as a promising therapeutic target in the context of chronic inflammation

Directional Growth of Human Neuronal Axons in a Microfluidic Device with Nanotopography on Azobenzene-Based Material

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 microfluidic device. In the axonal compartment, the photoinscribed nanotopography on a thin film of azobenzene-containing molecular glass efficiently guides the growth of axons. Integration of nanotopographic patterns with a compartmentalized microfluidic chip creates a human neuron-based model that supports superior axonal alignment in an isolated microenvironment. The practical utility of the chip by studying oxygen-glucose deprivation-induced damage for the isolated and aligned axons is demonstrated here. The created chip model represents a sophisticated platform and a novel tool for enhanced and long-term axon-targeted in vitro studies.publishedVersionPeer reviewe

Spatial Variability of Shear Wave Velocity Using Geostatistical Analysis in Mashhad City, NE Iran

Objective: To determine the frequency of mutations known to cause autosomal dominant Parkinson disease (PD) in a series with more than 10% of Sweden's estimated number of PD patients. Methods: The Swedish Parkinson Disease Genetics Network was formed as a national multicenter consortium of clinical researchers who together have access to DNA from a total of 2,206 PD patients; 85.4% were from population-based studies. Samples were analyzed centrally for known pathogenic mutations in SNCA (duplications/triplications, p.Ala30Pro, p.Ala53Thr) and LRRK2 (p.Asn1437His, p.Arg1441His, p.Tyr1699Cys, p.Gly2019Ser, p.Ile2020Thr). We compared the frequency of these mutations in Swedish patients with published PD series and the gnomAD database. Results: A family history of PD in first- and/or second-degree relatives was reported by 21.6% of participants. Twelve patients (0.54%) carried LRRK2 p.(Gly2019Ser) mutations, one patient (0.045%) an SNCA duplication. The frequency of LRRK2 p.(Gly2019Ser) carriers was 0.11% in a matched Swedish control cohort and a similar 0.098% in total gnomAD, but there was a marked difference between ethnicities in gnomAD, with 42-fold higher frequency among Ashkenazi Jews than all others combined. Conclusions: In relative terms, the LRRK2 p.(Gly2019Ser) variant is the most frequent mutation among Swedish or international PD patients, and in gnomAD. SNCA duplications were the second most common of the mutations examined. In absolute terms, however, these known pathogenic variants in dominant PD genes are generally very rare and can only explain a minute fraction of familial aggregation of PD. Additional genetic and environmental mechanisms may explain the frequent co-occurrence of PD in close relatives