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Spinal cord injury continues to be, unarguably, a largely convoluted challenge of modern medicine, leaving millions of patients worldwide with devastating lifetime consequences. Currently, there are not medical treatments capable of efficiently and completely restoring lost autonomous, motor and/or sensory functions. In this context, along with the development of a next generation of biomedical platforms, the inclusion of graphene-based materials into neuroprotective and neuroregenerative strategies has potential to boost the structural and physiological recovery of the injured spinal cord. Particularly, these nanomaterials are updating important principles of tissue engineering concerning the development of biocompatible scaffolds suitable for replacing damaged neural microenvironments in a near-future scenario. In this work, a new design concept is used to fabricate graphene composites with fibrous-porous architectures able to support enhanced interactions with neural cells. In detail, electrospun nanofibres were accommodated onto the surface of reduced graphene oxide sheets to generate combinatorial 3D systems. The physiochemical and biological features of fibrous-porous constructs were associated with the properties of their nanofibrous coatings, resulting, consequently, in unique microenvironments capable of supporting highly viable, mature and interconnected neural circuits in vitro. Due to their enhanced performance, these graphene composites were further adapted to recreate the gray matter of the spinal cord, becoming a key component of a new biomimetic scaffolding methodology. In this way, for ensuring a spatial organization resembling the native spinal cord, where the center of gray matter is surrounded by white matter, the selected fibrous-porous graphene composite was incorporated within a 3D nanofibrous framework. Furthermore, specific regions of this biomimetic framework were loaded with polycaprolactone-chitosan nanofibres and polycaprolactone-graphene microfibers with the purpose of mimicking the white matter. After in vitro and ex vivo tests, the final biomimetic scaffold was then implanted into hemisected rats for 4 months, after which it was possible to observe promising neuroprotective and neuroregenerative features together with encouraging signs of forelimb function recovery. Based on these findings, the reported biomimetic graphene-based scaffolds hold potential to integrate advanced therapeutic strategies for countering spinal cord injury. A lesão da medula espinal continua a ser indiscutivelmente um grande desafio da medicina moderna, provocando efeitos devastadores e permanentes a milhões de pacientes por todo o mundo. Não existem atualmente tratamentos médicos capazes de restaurar, de uma forma eficaz e completa, as funções sensoriais, motoras e/ou autónomas danificadas/perdidas depois desta lesão. Neste contexto, e seguindo o desenvolvimento da próxima geração de plataformas biomédicas, a inclusão de grafeno e materiais derivados em estratégias de neuroproteção e neurorregeneração tem mostrado potencial para estimular a recuperação estrutural e fisiológica do tecido medular lesionado. Particularmente, estes nanomateriais têm impulsionado princípios importantes da engenharia de tecidos aplicados no desenvolvimento de suportes celulares biocompatíveis denominados scaffolds, com o intuito de garantir, num futuro próximo, a substituição de microambientes neurais danificados. Neste trabalho é apresentada e validada uma nova metodologia para fabricar compósitos de grafeno com uma arquitetura fibrosa-porosa apta a promover interações otimizadas com células neurais. Em detalhe, nanofibras poliméricas fabricadas através de eletrofiação foram distribuídas pela superfície de folhas de óxido de grafeno reduzido com o intuito de gerar sistemas combinatórios 3D. Foi demonstrado que as propriedades biológicas e físico-químicas das construções fibrosas-porosas dependiam das características do revestimento nanofibroso utilizado, o que resultou, consequentemente, em scaffolds únicos capazes de acomodar circuitos neurais maduros altamente viáveis e interconectados in vitro. A performance auspiciosa destes compósitos de grafeno levou à sua adaptação com o objetivo de recriar a matéria cinzenta da medula espinal numa nova estratégia biomimética de engenharia de tecido neural. Para assegurar a organização espacial característica da medula, onde o centro de matéria cinzenta está rodeado por matéria branca, o compósito fibroso-poroso de grafeno selecionado foi incorporado no interior de um sistema nanofibroso 3D. Nesta construção biomimética, com vista à simulação da matéria branca, foi também integrado um sistema formado por nanofibras de policaprolactona e quitosano e microfibras de policaprolactona e grafeno. Depois de testado in vitro e ex vivo, o scaffold final foi implantado em ratazanas paralisadas com uma hemissecção cervical e, após 4 meses, foi possível observar impactos positivos na neuroproteção e neurorregeneração do tecido medular. Desta forma, e também considerando os resultados promissores associados à recuperação comportamental dos animais intervencionados, é possível constatar o potencial destes scaffolds de grafeno em integrar estratégias terapêuticas avançadas suscetíveis de contrariar os efeitos adversos da lesão da medula espinal. Programa Doutoral em Nanociências e Nanotecnologia

MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells' function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating β-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together., This research was supported by the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain) and Co-financed by the European Union (FEDER) “A way to make Europe” (project references SBPLY/17/000376 and SBPLY/21/180501/000097) and by the Ministerio de Ciencia, Innovación y Universidades (RTI2018-096328-B-I00). Altea Soto was funded by the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain) M. Asunción Barreda-Manso is funded by the Council of Health of the Regional Government of Castilla La Mancha (Spain), through: “Convocatoria de Ayudas Regionales a la Investigación en Biomedicina y Ciencias de la Salud” (II-2020_05). Irene Novillo Algaba is funded by the Next Generation Funds of the European Union through the “Programa Investigo”.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain–Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood–brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml−1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

Duloxetine is a clinical drug that is primarily used for treatment of depression and pain, but it has side effects of addiction and tolerance. Cytochrome P450 (CYP) is its metabolic enzyme, and the drug’s biofunction results from its neuro-protective effect in animal and cell models. We aimed to investigate the duloxetine-induced neural cytotoxicity effect and its performance in an N2a cell neurite outgrowth model. Cell death was assessed as cell viability using a Cell Count Kit-8 and further evaluated using bright-field images, propidium iodide (PI) and annexin V staining, colony-formation analysis, TUNEL staining of the cells, and biochemical testing. N2a cells were committed to differentiation by serum withdrawal and RA induction, and the neurite outgrowth was evaluated as the number of differentiated cells, longest neurite length, and average neurite length. Cell cycle analysis, PI and annexin V staining, mRNA expression, and biochemical testing were used to evaluate the drug effects on differentiation. The induction of neural cell death by duloxetine was not affected by classic cell death inhibitors but was promoted by the CYP inducer rifampicin. N2a cell neurite outgrowth was promoted by duloxetine via reduction of the CYP2D6 and MDA levels and induction of Bdnf protein levels. Duloxetine induces neural cell death through effects on CYP and promotes N2a cell neurite outgrowth by regulating CYP, Bdnf protein, and the intracellular lipid peroxidation level.

International audience; Investigation of the toxicity triggered by chemicals on the human brain has traditionally relied on approaches using rodent in vivo models and in vitro cell models including primary neuronal cultures and cell lines from rodents. The issues of species differences between humans and rodents, the animal ethical concerns and the time and cost required for neurotoxicity studies on in vivo animal models, do limit the use of animal-based models in neurotoxicology. In this context, human cell models appear relevant in elucidating cellular and molecular impacts of neurotoxicants and facilitating prioritization of in vivo testing.The SH-SY5Y human neuroblastoma cell line (ATCC® CRL-2266TM) is one of the most used cell lines in neurosciences, either undifferentiated or differentiated into neuron-like cells. This review presents the characteristics of the SH-SY5Y cell line and proposes the results of a systematic review of literature on the use of this in vitro cell model for neurotoxicity research by focusing on organic environmental pollutants including pesticides, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), flame retardants, PFASs, parabens, bisphenols, phthalates, and PAHs. Organic environmental pollutants are widely present in the environment and increasingly known to cause clinical neurotoxic effects during fetal & child development and adulthood. Their effects on cultured SH-SY5Y cells include autophagy, cell death (apoptosis, pyroptosis, necroptosis, or necrosis), increased oxidative stress, mitochondrial dysfunction, disruption of neurotransmitter homeostasis, and alteration of neuritic length. Finally, the inherent advantages and limitations of the SH-SY5Y cell model are discussed in the context of chemical testing.

Thesis (PhD(Medical Science)--University of South Australia, 2022. Includes bibliographical references (pages 138-146) Neural cells hold promising potentials for treating neurological disorders. However, their scarce sources have limited their clinical application. Cell reprogramming with small molecules provides an opportunity to generate neural cells from easily obtainable somatic cells, such as fibroblasts and urine-derived cells, in a safe and efficient manner. In this study, we have generated a small molecule aided protocol that can induce human skin fibroblasts into induced neural stem cells (iNSCs) and have generated another small molecule aided protocol that can induce human urine-derived cells into induced neurons (iNs). The results have shown that the induced cells possess neural stem cell-like and neuron-like characters, respectively.

The proposed thesis deals with the various aspects of tick-borne encephalitis virus infection in the host and the vector on the cellular level. It uncovers transcriptomic and proteomic responses in infected cells in the human neurons and astrocytes, and vector cells. It identifies the subgenomic flaviviral RNA as an important pathogenesis effector that can interfere with the vector RNAi pathway, and at the same time denotes the components of this pathway. It also describes the phenomenon of impairment of host protein and rRNA synthesis upon TBEV infection. Moreover, it uncovers the importance of quasispecies in the adaptation to vector and host cells.

Accumulating scientific and clinical evidence highlighted pathological hyperinflammation as a cardinal feature of SARS-CoV-2 infection and acute COVID-19 disease. With the emergence of long COVID-19 syndrome, several chronic health consequences, including neuropsychiatric sequelae, have gained attention from the public and medical communities. Since inflammatory mediators have also been accredited as putative biomarkers of suicidal ideations and behaviors, hyper- and neuroinflammation might share some colliding points, overlapping and being interconnected in the context of COVID-19. This review aims to provide a summary of current knowledge on the molecular and cellular mechanisms of COVID-19-associated hyper/neuroinflammation with focus on their relevance to the inflammatory hypothesis of suicide development. Subsequently, strategies to alleviate COVID-19 hyper/neuroinflammation by immunomodulatory agents (many of which at experimental stages) as well as psychopharmacologic/psychotherapeutic approaches are also mentioned. While suicide risk in COVID-19 survivors - until now little known - needs further analysis through longitudinal studies, current observations and mechanistic postulates warrant additional attention to this possibly emerging mental health concern.

Despite a major interest in understanding how the endothelial cell phenotype is established, the underlying molecular basis of this process is not yet fully understood. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells and differentiation of the resulting HiPS back to endothelial cells (Ec-Diff), as well as neural (Nn-Diff) cell lineage that contained both neurons and astrocytes. Furthermore, the identities of these cell lineages were established by gene array analysis. Here, we explored the same arrays to gain insight into the gene alteration processes that accompany the establishment of endothelial vs. non-endothelial neural cell phenotypes. We compared the expression of genes that code for transcription factors and epigenetic regulators when HiPS is differentiated into these endothelial and non-endothelial lineages. Our in silico analyses have identified cohorts of genes that are similarly up- or downregulated in both lineages, as well as those that exhibit lineage-specific alterations. Based on these results, we propose that genes that are similarly altered in both lineages participate in priming the stem cell for differentiation in a lineage-independent manner, whereas those that are differentially altered in endothelial compared to neural cells participate in a lineage-specific differentiation process. Specific GATA family members and their cofactors and epigenetic regulators (DNMT3B, PRDM14, HELLS) with a major role in regulating DNA methylation were among participants in priming HiPS for lineage-independent differentiation. In addition, we identified distinct cohorts of transcription factors and epigenetic regulators whose alterations correlated specifically with the establishment of endothelial vs. non-endothelial neural lineages.

Methamphetamine (METH) is a potent stimulant that induces a euphoric state but also causes cognitive impairment, neurotoxicity and neurodevelopmental deficits. Yet, the molecular mechanisms by which METH causes neurodevelopmental defects have remained elusive. Here we utilized human cerebral organoids and single-cell RNA sequencing (scRNA-seq) to study the effects of prenatal METH exposure on fetal brain development. We analyzed 20,758 cells from eight untreated and six METH-treated cerebral organoids and found that the organoids developed from embryonic stem cells contained a diverse array of glial and neuronal cell types. We further identified transcriptionally distinct populations of astrocytes and oligodendrocytes within cerebral organoids. Treatment of organoids with METH-induced marked changes in transcription in multiple cell types, including astrocytes and neural progenitor cells. METH also elicited novel astrocyte-specific gene expression networks regulating responses to cytokines, and inflammasome. Moreover, upregulation of immediate early genes, complement factors, apoptosis, and immune response genes suggests a neuroinflammatory program induced by METH regulating neural stem cell proliferation, differentiation, and cell death. Finally, we observed marked METH-induced changes in neuroinflammatory and cytokine gene expression at the RNA and protein levels. Our data suggest that human cerebral organoids represent a model system to study drug-induced neuroinflammation at single-cell resolution.

Patients with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection manifest mainly respiratory symptoms. However, clinical observations frequently identified neurological symptoms and neuropsychiatric disorders related to COVID‐19 (Neuro‐SARS2). Accumulated robust evidence indicates that Neuro‐SARS2 may play an important role in aggravating the disease severity and mortality. Understanding the neuropathogenesis and cellular mechanisms underlying Neuro‐SARS2 is crucial for both basic research and clinical practice to establish effective strategies for early detection/diagnosis, prevention, and treatment. In this review, we comprehensively examine current evidence of SARS‐CoV‐2 infection in various neural cells including neurons, microglia/macrophages, astrocytes, pericytes/endothelial cells, ependymocytes/choroid epithelial cells, and neural stem/progenitor cells. Although significant progress has been made in studying Neuro‐SARS2, much remains to be learned about the neuroinvasive routes (transneuronal and hematogenous) of the virus and the cellular/molecular mechanisms underlying the development/progression of this disease. Future and ongoing studies require the establishment of more clinically relevant and suitable neural cell models using human induced pluripotent stem cells, brain organoids, and postmortem specimens.

The central nervous system (CNS) is the most complex structure in the body, consisting of multiple cell types with distinct morphology and function. Development of the neuronal circuit and its function rely on a continuous crosstalk between neurons and non-neural cells. It has been widely accepted that extracellular vesicles (EVs), mainly exosomes, are effective entities responsible for intercellular CNS communication. They contain membrane and cytoplasmic proteins, lipids, non-coding RNAs, microRNAs and mRNAs. Their cargo modulates gene and protein expression in recipient cells. Several lines of evidence indicate that EVs play a role in modifying signal transduction with subsequent physiological changes in neurogenesis, gliogenesis, synaptogenesis and network circuit formation and activity, as well as synaptic pruning and myelination. Several studies demonstrate that neural and non-neural EVs play an important role in physiological and pathological neurodevelopment. The present review discusses the role of EVs in various neurodevelopmental disorders and the prospects of using EVs as disease biomarkers and therapeutics.

Ischemic stroke caused by cerebral artery occlusion induces neurological deficits because of cell damage or death in the central nervous system. Given the recent therapeutic advances in reperfusion therapies, some patients can now recover from an ischemic stroke with no sequelae. Currently, reperfusion therapies focus on rescuing neural lineage cells that survive in spite of decreases in cerebral blood flow. However, vascular lineage cells are known to be more resistant to ischemia/hypoxia than neural lineage cells. This indicates that ischemic areas of the brain experience neural cell death but without vascular cell death. Emerging evidence suggests that if a vascular cell-mediated healing system is present within ischemic areas following reperfusion, the therapeutic time window can be extended for patients with stroke. In this review, we present our comments on this subject based upon recent findings from lethal ischemia following reperfusion in a mouse model of stroke.

The field of bioelectrochemistry is growing rapidly and is beginning to have a significant impact on the practice of medicine and biology. Due to its potency, electrochemical impedance spectroscopy (EIS) is used frequently by the scientists working at the interface between biology, medicine and electrochemistry. The aim of this paper is to describe the current state of the art devoted to the application of EIS in neural science, with the particular focus on the application of the impedance measurements to monitor cell adhesion, cell growth and morphological changes during neuronal differentiation, to track cell apoptosis, neural degeneration, and to distinguish among specific types of cells. These applications are investigated in terms of the electrochemical behaviour of cells and tissues, including the description of equivalent circuits used for modelling the electrode-tissue interface. Therefore, this paper combines the potential applications of EIS in neural science with the electrochemical background necessary for the proper interpretation of acquired results.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a major cellular energy sensor that is activated by an increase in the AMP/adenosine triphosphate (ATP) ratio. This causes the initiation of adaptive cellular programs, leading to the inhibition of anabolic pathways and increasing ATP synthesis. AMPK indirectly inhibits mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a serine/threonine kinase and central regulator of cell growth and metabolism, which integrates various growth inhibitory signals, such as the depletion of glucose, amino acids, ATP and oxygen. While neuroprotective approaches by definition focus on neurons, that are more sensitive under cell stress conditions, astrocytes play an important role in the cerebral energy homeostasis during ischemia. Therefore, the protection of astrocytic cells or other glial cells may contribute to the preservation of neuronal integrity and function. In the present study, it was thus hypothesized that a preventive induction of energy deprivation-activated signaling pathways via AMPK may protect astrocytes from hypoxia and glucose deprivation. Hypoxia-induced cell death was measured in a paradigm of hypoxia and partial glucose deprivation in vitro in the immortalized human astrocytic cell line SVG. Both the glycolysis inhibitor 2-deoxy-d-glucose (2DG) and the AMPK activator A-769662 induced the phosphorylation of AMPK, resulting in mTORC1 inhibition, as evidenced by a decrease in the phosphorylation of the target ribosomal protein S6 (RPS6). Treatment with both 2DG and A-769662 also decreased glucose consumption and lactate production. Furthermore, A-769662, but not 2DG induced an increase in oxygen consumption, possibly indicating a more efficient glucose utilization through oxidative phosphorylation. Hypoxia-induced cell death was profoundly reduced by treatment with 2DG or A-769662. On the whole, the findings of the present study demonstrate, that AMPK activation via 2DG or A-769662 protects astrocytes under hypoxic and glucose-depleted conditions.

Peptide aggregation into oligomers and fibrillar architectures is a hallmark of severe neurodegenerative pathologies, diabetes mellitus or systemic amyloidoses. The polymorphism of amyloid forms and their distribution are both effectors that potentially modulate the disease, thus it is important to understand the molecular basis of protein amyloid disorders through the interaction of the different amyloid forms with neural cells and tissues. Here we explore the effect of amyloid fibrils on the human neuroblastoma (SH-SY5Y) cell line in vitro. We control the kinetic of fibrillization of insulin at low pH and higher temperature. We use a multiscale characterization via fluorescence microscopy and multimodal scanning probe microscopy to correlate the number of cells and their morphology, with the finer details of the insulin deposits. Our results show that insulin aggregates deposited on neuroblastoma cell cultures lead to a progressive modification and decreased number of cells that correlates with the degree of fibrillization. SPM unravels that the aggregates strongly interact with the cell membrane, forming a stiff encase that possibly leads to an increased cell membrane stiffness and deficit in the metabolic exchanges between the cells and their environment. The presence of fibrils does not affect the number of cells at 24h whereas drop down to 60% is observed after 48h of incubation.

(‒)-Cannabidiol (CBD) is one of the major phytocannabinoids extracted from the Cannabis genus. Its non-psychoactiveness and therapeutic potential, partly along with some anecdotal—if not scientific or clinical—evidence on the prevention and treatment of neurological diseases, have led researchers to investigate the biochemical actions of CBD on neural cells. This review summarizes the previously reported mechanistic studies of the CBD actions on primary neural cells at the in vitro cell-culture level. The neural cells are classified into neurons, microglia, astrocytes, oligodendrocytes, and neural stem cells, and the CBD effects on each cell type are described. After brief introduction on CBD and in vitro studies of CBD actions on neural cells, the neuroprotective capability of CBD on primary neurons with the suggested operating actions is discussed, followed by the reported CBD actions on glia and the CBD-induced regeneration from neural stem cells. A summary section gives a general overview of the biochemical actions of CBD on neural cells, with a future perspective. This review will provide a basic and fundamental, but crucial, insight on the mechanistic understanding of CBD actions on neural cells in the brain, at the molecular level, and the therapeutic potential of CBD in the prevention and treatment of neurological diseases, although to date, there seem to have been relatively limited research activities and reports on the cell culture-level, in vitro studies of CBD effects on primary neural cells.

Cancer is a complex disease without a unified explanation for its cause so far. Our recent work demonstrates that cancer cells share similar regulatory networks and characteristics with embryonic neural cells. Based on the study, I will address the relationship between tumor and neural cells in more details. I collected the evidence from various aspects of cancer development in many other studies, and integrated the information from studies on cancer cell properties, cell fate specification during embryonic development and evolution. Synthesis of the information strongly supports that cancer cells share much more similarities with neural progenitor/stem cells than with mesenchymal-type cells and that tumorigenesis represents a process of gradual loss of cell or lineage identity and gain of characteristics of neural cells. I also discuss cancer EMT, a concept having been under intense debate, and possibly the true meaning of EMT in cancer initiation and development. This synthesis provides fresh insights into a unified explanation for and a previously unrecognized nature of tumorigenesis, which might not be revealed by studies on individual molecular events. The review will also present some brief suggestions for cancer research based on the proposed model of tumorigenesis.

Cell replacement therapy in the nervous system has a rich history, with ∼40 years of research and ∼30 years of clinical experience. There is compelling evidence that appropriate cells can integrate and function in the dysfunctioning human nervous system, but the clinical results are mixed in practice. A number of factors conspire to vary patient outcome: the indication, cell source, patient selection, and team performing transplantation are all variables that can affect efficacy. Most early clinical trials have used fetal cells, a limited cell source that resists scale and standardization. Direct fetal cell transplantation creates significant challenges to commercialization that is the ultimate goal of an effective cell therapy. One approach to help scale and standardize fetal cell preparations is the expansion of neural cells in vitro. Expansion is achieved by transformation or through the application of mitogens before cryopreservation. Recently, neural cells derived from pluripotent stem cells have provided a scalable alternative. Pluripotent stem cells are desirable for manufacturing but present alternative concerns and manufacturing obstacles. All cell sources require robust and reproducible manufacturing to make nervous system cell replacement therapy an option for patients. Here, we discuss the challenges and opportunities for cell replacement in the nervous system. In this review, we give an overview of completed and ongoing neural cell transplantation clinical trials, and we discuss the challenges and opportunities for future cell replacement trials with a particular focus on pluripotent stem cell-derived therapies.

[ES] La problemática asociada a las patologías del sistema nervioso viene del limitado potencial de regeneración de los tejidos nerviosos, y especialmente del SNC. Las lesiones en estos tejidos suponen un importante problema de salud pública pues su incidencia y prevalencia son muy elevadas, dado que el tejido no se recupera y la lesión es de por vida. Esto supone un coste económico elevado para el sistema sanitario y una baja autoestima por parte del paciente lesionado. En este contexto y para solventar este tipo de lesiones, ha surgido y cobra cada vez más importancia, la ingeniería tisular. Para el sistema nervioso, una aproximación consiste en combinar neuronas y células de soporte en un biomaterial electroconductor que permita la estimulación eléctrica, ya que este estímulo físico es necesario para el correcto desarrollo, funcionamiento y reparación del sistema nervioso. El presente Trabajo de Fin de Grado se enmarca en este campo de estudio: la regeneración axonal empleando corrientes eléctricas constantes para favorecer la producción de señales de regeneración nerviosa como la secreción de proteínas por parte de las células de soporte y guiar la extensión de las neuritas. Para ello, se ha desarrollado y obtenido un dispositivo para el cultivo celular in vitro que permite la integración de fibras guía de Ácido poli-L-láctico (PLA) recubiertas con biomateriales electroconductores como polipirrol (PPy) y oro que pueden acoplarse a un generador de corriente eléctrica. En estas fibras se cultivan células de soporte como células de Schwann (SCs) y neuronas procedentes del ganglio de la raíz dorsal (DRG). Este enfoque, a diferencia de emplear estimulación in vivo, está basada en promover la regeneración nerviosa tras una lesión mediante el trasplante de tractos axonales obtenidos in vitro empleando ingeniería tisular., [EN] Several issues associated to nervous system pathologies come from the limited potential for regeneration of nervous tissues, especially the CNS. Injuries to these tissues portray a major public health problem since their incidence and prevalence are very high, given that the tissue does not recover and the injury can affect the whole lifespan of the patient. This entails a high economic cost for the sanitary system and a low self-esteem for the injured person. In this context and to solve this type of injury, tissue engineering has emerged and is becoming increasingly important. For the nervous system, one approach is to combine neurons and supporting cells in an electroconductive biomaterial that allows electrical stimulation, since this physical stimulus is necessary for the proper development, function, and repair of the nervous system. The present End-of-Degree Project is enclosed within this field of study: axonal regeneration using constant electric currents to enhance the production of nerve regeneration signals such as secretion of proteins by supporting cells and guide neurite extension. To this end, a device for in vitro cell culture has been developed and obtained which allows the integration of guidance fibers of poly-L-lactic acid (PLA) coated with electroconductive biomaterials such as polypyrrole (PPy) and gold that can be coupled to an electric current generator. Supporting cells such as Schwann cells (SCs) and dorsal root ganglion neurons (DRG) are grown on these fibers. This approach, unlike utilizing stimulation in vivo, is based on promoting nerve regeneration after injury by transplanting axonal tracts obtained in vitro using tissue engineering.

Human dental pulp stem cells (hDPSCs) are promising source of cells for numerous and varied regenerative medicine applications as those possess high proliferation potential with multilineage differentiation capacity compare to other sources of adult stem cells; therefore, hDPSCs could be the good source for autologous transplantation in tissue engineering and regenerative medicine. In this study stem cells were isolated from dental pulp and were characterised by flowcytometry and immunocytochemistry. The controlled cells as well as, 7-day cultured cells were positive for transcription factors, OCT 4 and SOX 2 thatconfirmed isolated cellsasmesenchymal stem cells (MSCs). These cells showed positive expression for CD 19, CD 73, CD 90, CD 105 and are negative for CD 34, CD 45. Viability of hDPSCS were studied by trypan blue (TB) staining and fluorescent microscopic study. After 7 days of passaging by using several growth factors, cells express neural cell markers oligodendrocyte and glial fibrillary acidic protein. Specifically, osteocytes were grown from dental pulp MSCSsin vitro with the help of growth factors, dexamethasone, ascorbic acid-2- phosphate and β-glycerophosphate whereas, adipocytes were grown with indomethacin, 3-isobutyl-1-methylxanthine and insulin. Osteocytes and adipocytes were characterized by von Kossa and Oil red O staining, respectively. Chromosomal analysis of dental pulp-MSCs was done for qualitative assessment of MSCs. Karyotyping indicated diploid chromosome number in dental pulp derived MSCs. In vitro grown osteocytes could be used for bone fracture reunion cases, and adipocytes could be used for further research purposes., Biological sciences; Cell biology; Stem cells research; Biotechnology; Health sciences; Dentistry; Dental pulp; Stem cells; Neural cells; Flow cytometry; Immunocytochemistry.

Enterovirus 71 (EV71) can invade the central nervous system (CNS) and cause neurological disease. Accumulating evidence indicates that EV71 can directly infect neurons in the CNS. Innate immune responses in the CNS have been known to play an essential role in limiting pathogen infections. Thus, investigating the effects of EV71 infection of neural cells is important for understanding disease pathogenesis. In this study, human neural cells were infected with EV71, and interferon&beta, (IFN&beta, ) expression was examined. Our results show that IFN&beta, expression was upregulated in EV71-infected neural cells via pattern recognition receptors (PRRs) sensing of virus RNA. The PRRs Toll-like receptor 3 (TLR3), Toll-like receptor 8 (TLR8), and melanoma differentiation-associated gene-5 (MDA-5), but not retinoic acid-inducible gene-I (RIG-I) and Toll-like receptor 7 (TLR7), were found to be EV71-mediated IFN&beta, induction. Although viral proteins exhibited the ability to cleave mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-&beta, (TRIF) in neural cells, levels of viral protein expression were low in these cells. Furthermore, neural cells efficiently produced IFN&beta, transcripts upon EV71 vRNA stimulation. Treating infected cells with anti-IFN&beta, antibodies resulted in increased virus replication, indicating that IFN&beta, release may play a role in limiting viral growth. These results indicate that EV71 infection can induce IFN&beta, expression in neural cells through PRR pathways.

The limited access to functional human brain tissue has led to the development of stem cell-based alternative models. The differentiation of human pluripotent stem cells into cerebral organoids with self-organized architecture has created novel opportunities to study the early stages of the human cerebral formation. Here we applied state-of-the-art label-free shotgun proteomics to compare the proteome of stem cell-derived cerebral organoids to the human fetal brain. We identified 3,073 proteins associated with different developmental stages, from neural progenitors to neurons, astrocytes, or oligodendrocytes. The major protein groups are associated with neurogenesis, axon guidance, synaptogenesis, and cortical brain development. Glial cell proteins related to cell growth and maintenance, energy metabolism, cell communication, and signaling were also described. Our data support the variety of cells and neural network functional pathways observed within cell-derived cerebral organoids, confirming their usefulness as an alternative model. The characterization of brain organoid proteome is key to explore, in a dish, atypical and disrupted processes during brain development or neurodevelopmental, neurodegenerative, and neuropsychiatric diseases. ispartof: FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY vol:7 ispartof: location:Switzerland status: published

We have developed a concept of hybrid carbon nanomaterials, where different allotropes of carbon are integrated into a structure. In order to facilitate the long-term measurements in vivo, the cellular response at the bioelectric interface should be optimized. Indeed, failure of implant integration has been proposed to be the main reason for sensor failure in vivo. Most strategies to enhance electrode integration into target tissue exploit a protective layer or barrier on an electrode substrate. For the detection of neurotransmitters, this is not as suitable strategy, because (1) such films give rise to an increased background electrode capacitance and impedance, and (2) act as a diffusion barrier and as a result, a decreased amount of the analyte reaches the electrode surface and the kinetics is compromised. Here we demonstrate that we can regulate the cellular response just with the electrode material. Specifically, we will show that it is possible to combine the properties of different carbon allotropes toobtain hybrid materials with enhanced neural response. We will present three examples of the approach: (i) functionalized nanodiamonds on tetrahedral amorphous carbon (ta-C), (ii) multi-walled carbon nanotubes grown directly on top of to-C, and (iii) carbon nanofibres synthesized on top of ta-C thin films. We demonstrate that hybrid structures may promote neural integration as, for example, hydrogen-terminated nanodiamonds enhance neural cell viability and while not increasing glial cell viability. Moreover, carbon nanofibers show prominence for tuning the cellular response as their dimension match biologically relevant cues. We show that nanofiber dimensions significantly alter glial and neural cell adhesion as well as their morphology. The properties of the hybrid structures can be tailored, both geometrically and chemically, with high definition. Consequently, these materials possess exceptionally high potential to achieve optimal host response just with the electrode material.

Spinal cord injury is a devastating neurological disorder that damages motor, sensory, and autonomic pathways. Recent advances in stem cell therapy have allowed for the in vitro generation of motor neurons showing electrophysiological and synaptic activity, expression of canonical motor neuron biomarkers, and the ability to graft into spinal lesions. Clinical translation, especially the transplantation of motor neuron precursors in spinal lesions, has thus far been elusive because of stem cell heterogeneity and protocol variability, as well as a hostile microenvironment such as inflammation and scarring, which yield inconsistent pre-clinical results without a consensus best-practice therapeutic strategy. Induced pluripotent stem cells (iPSCs) in particular have lower ethical and immunogenic concerns than other stem cells, which could make them more clinically applicable. In this review, we focus on the differentiation of iPSCs into neural precursors, motor neuron progenitors, mature motor neurons, and motor neuron subtype fates. Previous reviews have summarized motor neuron development and differentiation, but an up-to-date summary of technological and experimental advances holding promise for bench-to-bedside translation, especially those targeting individual motor neuron subtypes in spinal cord injury, is currently lacking. We discuss biological mechanisms of motor neuron lineage, recent experimental protocols and techniques for motor neuron differentiation from iPSCs, and transplantation of neural precursors and motor neuron lineage cells in spinal cord lesions to restore motor function. We emphasize efficient, clinically safe, and personalized strategies for the application of motor neurons and their subtypes as therapy in spinal lesions.

Background Very few studies have identified receptor molecules for dengue virus (DENV) on neural cells. This study was designed to identify putative receptor/(s) involved in entry of DENV-3 in human neural cells of various lineages; neuronal-SH-SY5Y, astroglial-U-87 MG and microglial-CHME-3 cells. Result Virus overlay protein binding assay, LC-MS/MS and SEQUEST identified prohibitin1/2 (PHB1/2) as interacting proteins on SH-SY5Y, CHME-3, and U-87 MG cells. Infection inhibition and siRNA assays confirmed the role of PHB1/2 in the entry of DENV-3 into SH-SY5Y and CHME-3 cells but not in U-87 MG cells. Indirect immunofluorescence and flow-cytometry demonstrated the presence of PHB1/2 on the surface of SH-SY5Y and CHME-3 cells. Co-immunoprecipitation and Western blot, as well as double labelling, reconfirmed the interaction between PHB1/2 and DENV-3 EDIII protein. Conclusion These observations together for the first time indicate that PHB1/2 may serve as a putative receptor for DENV-3 in SH-SY5Y and CHME-3 cells. The study provided insights into DENV-3 and neural cell interactions.

Introduction: Endometrial stem cells (EnSCs) were identified for the first time in 2004. These cells are capable of extensive self-renewal and have the potency to differentiate into chondrocyte, osteocyte, adipocyte, neuron and oligodendrocyte. PI3K/Akt signaling has been implicated in multiple cellular and organ functions, including differentiation, survival and cell death and its inhibition leads to cell differentiation. The purpose of this study was to investigate the differentiation of endometrial stem cells into neural cells by inhibition of PI3K/Akt pathway using small molecule Ly294002. Materials and Methods: Endometrial tissues were treated enzymatically and segregated cells were cultured in DMEM/F12 with 10% FBS. The flow cytometry analysis was perfomed for CD105, CD90, CD146, CD31 and CD34 at the third passage. Then the neurogenic differentiation was evaluated at the third passage, 21 days after induction with differentiation media. Immunocytochemistry and Real-time PCR were performed to investigate the expression of specific neural stem cells markers. Results: The flow cytometry analysis showed that EnSCs were positive for CD90, CD105 and CD146 and negative for CD31 and CD34. Immunocytochemistry showed that the expression of nestin, NF and Chat neuronal markers in the cells treated with small molecule Ly294002. Real-time PCR also indicated expression of NF and Chat neuronal markers at the mRNA level. Conclusion: According to the findings of this study it can be concluded that the EnSCs have neural differentiation potency in the suitable differentiation milieu. Ly294002 small molecules by inhibiting PI3K / Akt pathway possibly can prevent cell proliferation and induce cell differentiation

Qi Qi,1,2 Yuxin Zhang,1 Lin Shen,1 Rui Wang,1 Jiansheng Zhou,1 Hezuo Lü,1,3 Jianguo Hu1,3 1Anhui Key Laboratory of Tissue Transplantation, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, People’s Republic of China; 2Department of Histology and Embryology, Bengbu Medical College, Bengbu, Anhui, People’s Republic of China; 3Department of Clinical Laboratory, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, People’s Republic of China Purpose: Our purpose was to systematically investigate the expression pattern and role of Olig1 in neural cells during rat spinal cord development.Animals and methods: Spinal cord tissues were dissected from Sprague–Dawley rats at embryonic day 14.5 (E14.5) and E18.5, postnatal day 0 (P0), P3, P7, postnatal 2 weeks (P2W), P4W, and adults (more than 2 months after birth), respectively. The expression of Olig1 was determined by Western blot and immunostaining. To observe expression of Olig1 in different neural cell types, a double immunohistochemical staining was performed using antibodies against Olig1 with O4, ß-tubulin, glial fibrillary acidic protein (GFAP), and myelin basic protein, respectively.Results: The expression of Olig1 protein shows a significant level change in rat spinal cord at different developmental time points. Starting with E14.5, the expression gradually increased and peaked at E18.5. Olig1 decreased gradually from P3 and reached its lowest level on P7. However, interestingly, the Olig1 expression increased again from P2W, until adulthood. Olig1 was coexpressed with O4-positive oligodendrocyte progenitor cells (OPCs) and β-tubulin-positive neurons at all time points during development. Olig1 was also coexpressed transiently with GFAP-positive astrocytes at only E14.5. Olig1 was localized in the cytoplasm of O4- and β-tubulin-positive cells during the period from E14.5 to adult.Conclusion: The expression of Olig1 in OPCs and neurons at all time points during development and in astrocytes at E14.5 suggests that Olig1 may play an important role in the generation and maturation of specific neural cells during development of spinal cord. Our results contribute to understanding the mechanism underlying developmental regulation of neural cells by Olig1. Keywords: Olig1, spinal cord, development, neural cells

Spinal cord injury has long been a prominent challenge in the trauma repair process. Spinal cord injury is a research hotspot by virtue of its difficulty to treat and its escalating morbidity. Furthermore, spinal cord injury has a long period of disease progression and leads to complications that exert a lot of mental and economic pressure on patients. There are currently a large number of therapeutic strategies for treating spinal cord injury, which range from pharmacological and surgical methods to cell therapy and rehabilitation training. All of these strategies have positive effects in the course of spinal cord injury treatment. This review mainly discusses the problems regarding stem cell therapy for spinal cord injury, including the characteristics and action modes of all relevant cell types. Induced pluripotent stem cells, which represent a special kind of stem cell population, have gained impetus in cell therapy development because of a range of advantages. Induced pluripotent stem cells can be developed into the precursor cells of each neural cell type at the site of spinal cord injury, and have great potential for application in spinal cord injury therapy.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)., The central nervous system (CNS) is the most complex structure in the body, consisting of multiple cell types with distinct morphology and function. Development of the neuronal circuit and its function rely on a continuous crosstalk between neurons and non-neural cells. It has been widely accepted that extracellular vesicles (EVs), mainly exosomes, are effective entities responsible for intercellular CNS communication. They contain membrane and cytoplasmic proteins, lipids, non-coding RNAs, microRNAs and mRNAs. Their cargo modulates gene and protein expression in recipient cells. Several lines of evidence indicate that EVs play a role in modifying signal transduction with subsequent physiological changes in neurogenesis, gliogenesis, synaptogenesis and network circuit formation and activity, as well as synaptic pruning and myelination. Several studies demonstrate that neural and non-neural EVs play an important role in physiological and pathological neurodevelopment. The present review discusses the role of EVs in various neurodevelopmental disorders and the prospects of using EVs as disease biomarkers and therapeutics.

The formation of amyloid fibrils from soluble proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. Lysozyme was converted from its soluble native state into highly organized amyloid fibrils. Ultrasonic treatment was used to break amyloid fibrils to fibrillar fragments–seeds. Atomic force microscopy and fluorescence microscopy was employed to characterize the morphology of the amyloid assemblies and neural cells–amyloid complexes. Our results demonstrate that prefibrillar intermediated and their mixture with proteins exhibit toxicity, although native proteins and fibrils appear to have no effect on number of cells. Our findings confirm that innocuous hen lysozyme can be engineered to produce both cytotoxic fibrillar fragments and non-toxic mature amyloid fibrils. Our work further strengthens the claim that amyloid conformation, and not the identity of the protein, is key to cellular toxicity and the underlying specific cell death mechanism.

The proposed thesis focuses on the description of flavivirus-host interactions in case of tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). In more detail, the TBEV-induced host responses in human cells of neural origin and interferon-mediated protection were described together with the identification of a new phenomenon of TBEV-induced host transcriptional and translational shut-off. In addition, virus-derived molecules with hypothetical immunomodulatory characteristics, TuORF and ZIKV sfRNA, were analysed for their presence and possible function during the infection.

The aberrant expression of lncRNAs has been inferred to be closely related with the progression of neural ischemia/reperfusion (I/R) injury. RMRP is an lncRNA associated with I/R injury. In order to determine the role of RMRP in I/R injury, the effects of RMRP knockdown on oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced injury in SH-SY5Y cells were evaluated. The effect of OGD/R administration on the expression of RMRP and apoptosis in SH-SY5Y cells, and the effect of RMRP suppression by siRNA on the impairments of cells proliferation and mobility potential due to OGD/R administration were assessed in the current study. At the molecular level, the current study detected the expressions of indicators involved in autophagy and PI3K/Akt/mTOR-mediated apoptosis pathways. The OGD/R administration induced the expression of RMRP and apoptosis in SH-SY5Y cells. After RMRP knockdown, the proliferation potential of SH-SY5Y cells was restored, and apoptosis and cell cycle arrest were inhibited. Moreover, RMRP inhibition also increased the invasion and migration of SH-SY5Y cells which were treated with OGD/R. The effects of RMRP suppression on the phenotypes of SH-SY5Y were associated with the inhibition of LC3II, p-PI3K, p-Akt, and p-mTOR as well as the induction of P62 and Bcl-2. Inhibition of RMRP contributed to the improvement of OGD/R-induced neuronal injury, which might be mediated through the inhibition of autophagy and apoptosis pathways.

The function of human nervous system is critically dependent on proper interneuronal communication. Exosomes and other extracellular vesicles are emerging as a novel form of information exchange within the nervous system. Intraluminal vesicles within multivesicular bodies (MVBs) can be transported in neural cells anterogradely or retrogradely in order to be released into the extracellular space as exosomes. RNA loading into exosomes can be either via an interaction between RNA and the raft-like region of the MVB limiting membrane, or via an interaction between an RNA-binding protein-RNA complex with this raft-like region. Outflow of exosomes from neural cells and inflow of exosomes into neural cells presumably take place on a continuous basis. Exosomes can play both neuro-protective and neuro-toxic roles. In this review, we characterize the role of exosomes and microvesicles in normal nervous system function, and summarize evidence for defective signaling of these vesicles in disease pathogenesis of some neurodegenerative diseases.

Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...

DISP3 protein, also known as PTCHD2, belongs to the PTCHD family of proteins, which contain a sterol-sensing domain in their structure. The expression of the Disp3 gene is high in neural tissues and is regulated by thyroid hormone. The DISP3 gene is associated with development and progression of certain types of tumors, as well as with development of some neural pathologies. Neural stem cells also display high expression of the Disp3 gene. Neural stem cells are defined by their capability to self-renewal and capacity to differentiate into the basic types of neural cells - neurons, astrocytes, and oligodendrocytes. Precise regulation of the balance between proliferation and differentiation of neural stem cells is crucial for development of the central nervous system and its subsequent proper functioning, and disruption of this balance may lead to development of various pathologies. In this work we mainly focused on describing the function of the DISP3 protein in neural cells and tissues. We have shown that during differentiation of neural stem cells, the expression of the Disp3 gene is significant decreased. Furthermore, we have found that in neural stem and progenitor cells, the increased expression of the Disp3 gene promotes their proliferation. Moreover, when Disp3 expression was disrupted, the...

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations., Journal of Visualized Experiments. JoVE, 2017 (130), ISSN:1940-087X

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.

Damage to peripheral nerve tissue may cause loss of function in both the nerve and the targeted muscles it innervates. This study compared the repair capability of engineered nerve conduit (ENC), engineered fibroblast conduit (EFC), and autograft in a 10-mm tibial nerve gap. ENCs were fabricated utilizing primary fibroblasts and the nerve cells of rats on embryonic day 15 (E15). EFCs were fabricated utilizing primary fibroblasts only. Following a 12-week recovery, nerve repair was assessed by measuring contractile properties in the medial gastrocnemius muscle, distal motor nerve conduction velocity in the lateral gastrocnemius, and histology of muscle and nerve. The autografts, ENCs and EFCs reestablished 96%, 87% and 84% of native distal motor nerve conduction velocity in the lateral gastrocnemius, 100%, 44% and 44% of native specific force of medical gastrocnemius, and 63%, 61% and 67% of native medial gastrocnemius mass, respectively. Histology of the repaired nerve revealed large axons in the autograft, larger but fewer axons in the ENC repair, and many smaller axons in the EFC repair. Muscle histology revealed similar muscle fiber cross-sectional areas among autograft, ENC and EFC repairs. In conclusion, both ENCs and EFCs promoted nerve regeneration in a 10-mm tibial nerve gap repair, suggesting that the E15 rat nerve cells may not be necessary for nerve regeneration, and EFC alone can suffice for peripheral nerve injury repair.

Trabalho de Projeto do Mestrado Integrado em Engenharia Biomédica apresentado à Faculdade de Ciências e Tecnologia As superfícies rugosas surgiram como uma forma de alterar a superfície dos biomateriais e de aproximá-los mais do que é a verdadeira mimetização do ambiente biológico das células. Esta topografia aliada a uma química favorável à célula faz com que a probabilidade de sucesso do biomaterial seja maior. Neste contexto surgiu o tema desta dissertação que teve como principal objetivo desenvolver superfícies com diferentes topografias e estudar a sua influência no comportamento celular. A pulverização catódica r.f. magnetrão, foi a técnica utilizada para depositar filmes finos de carbono e poliamida 6,6 sobre PDMS e silício e consequentemente desenvolver topografias diferentes. Através da utilização do poli(dimetil siloxano) (PDMS) como substrato foi possível desenvolver superfícies com rugas a uma escala nanométrica e micrométrica, com um comprimento de onda entre 10 e 16 μm. Com a utilização do silício, a topografia formada foi apenas à escala nanométrica. O objetivo, ao desenvolver superfícies quimicamente iguais, mas com topografias diferentes, era a possibilidade de poder inferir sobre qual das características tem maior influência no comportamento da célula. As superfícies foram caracterizadas e posteriormente testadas in vitro como suporte para cultura de células do córtex de embrião de rato. Após 14 dias de contacto não se observou adesão celular nas superfícies com microrrugidade uma vez que os comprimentos de onda testados eram inferiores ao tamanho do corpo celular dos neurónios. Nas superfícies com nanorrugosidade ocorreu adesão celular e esta foi mais favorável nos filmes finos de carbono uma vez que estes apresentavam um potencial zeta menos negativo.Uma vez que o impedimento principal para a adesão das células à superfície com microrrugosidade foi o tamanho do corpo celular dos neurónios, não foi possível avaliar a orientação dos axónios. Com esse objetivo, a superfície com microrrugosidade foi colocada em contacto com uma rede neuronal já desenvolvida e ao fim de 14 dias observou-se que alguns axónios migraram para a superfície e continuaram a formar a rede neuronal. Este resultado vem mostrar que a superfície com microrrugosidade, apesar de não permitir a adesão inicial das células, apresenta uma química e uma topografia favorável para a posterior adesão de axónios. Com este trabalho deram-se os primeiros passos para a otimização de tubos guia que permitam a regeneração de axónios danificados do sistema nervoso periférico. Wrinkled surfaces have emerged as a way of modifying the surface of biomaterials and bringing them closer to the mimicking of the biological environment of cells. This topography, combined with a cell-friendly chemistry, makes the biomaterial's probability of success higher. It is in this context that the current dissertation was developed and its main objective was to produce wrinkled surfaces with different topographies and evaluate its influence on cell behavior.Sputtering r.f. magnetron was the technique used for the deposition of carbon and polyamide 6,6 thin films on polydimethylsiloxane (PDMS) and silicon surfaces and consequently develop different topographies. Though the use of PDMS as substrate, it was possible to develop surfaces with wrinkles at the nano- and micrometric scale, with a wavelength between 10 and 16 μm. With the use of silicon as substrate, the topography presented roughness only at the nanometric scale. The goal, by developing surfaces with the same chemistry but with different topographies, was to evaluate which of these two characteristic had the greatest influence on the cell behavior. The surfaces were characterized and then they were tested in vitro as support for rat embryo cortex cells. After 14 days of contact, no cell adhesion was observed on surfaces with microroughness, since their wavelength was smaller than the body size of the neurons. On the surfaces with nanoroughness, cell adhesion occurred and it was more favorable in the carbon thin films because the zeta potential on its surfaces was less negative. Since the main hindrance for cell adhesion to microroughness surfaces was the size of neurons’ cell body, it was not possible to evaluate the orientation of the axons through the topography. With this purpose, the wrinkled surface was placed in contact with an already developed neural network and, after 14 days of contact, it was observed that some axons migrate to the surface and continued to develop a neural network. This result shows that the surface with wrinkles at micrometric scale does not allow the initial cell adhesion, but presents a favorable chemistry and topography for the subsequent adhesion and growth of the neural network. With this work, the first steps were taken to optimize guide tubes for the regeneration of damaged axons of the peripheral nervous system. FCT

Background Tanshinone IIA is a key active ingredient of danshen, which is derived from the dried root or rhizome of Salviae miltiorrhizae Bge. The tanshinone IIA has protective effects against the focal cerebral ischemic injury. However, the underlying mechanisms remain unclear. Methods An in vitro model of cerebral ischemia was established by subjecting cultures of hippocampal neuronal cells to oxygen-glucose deprivation followed by reperfusion (OGD/R). The probes of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CMH2DCFDA) and 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine,iodide (JC-1) were used to determine the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production. Western-blot was used to detect the expression of proteins in HT-22 cells. Results The results of cell proliferative assays showed that the tanshinone IIA attenuated OGD/Rmediated neuronal cell death, with the evidence of increased cell viability. In addition, OGD/R exposure led to increase the levels of intracellular reactive oxygen species (ROS), which were significantly suppressed by tanshinone IIA treatment. Furthermore, tanshinone IIA treatment inhibited elevations in MMP and autophagy following exposure to OGD/R. Additionally, OGD/R promoted cell death with concomitant inhibiting phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of Rapamycin (mTOR) pathway, which was reversed by tanshinone IIA. Conclusion These results suggest that the tanshinone IIA protects against OGD/R-mediated cell death in HT-22 cells, in part, due to activating PI3K/Akt/mTOR pathway.

Recently, researchers have focused on the role of gut microbiota on human health and reported the existence of a bidirectional relationship between intestinal microbiota and the brain, referred to as microbiota-gut-brain axis (MGBA). In this context, the development of an organ-on-a-chip platform recapitulating the main players of the MGBA would help in the investigations of the biochemical mechanisms involved. In this work, we focused on the development of a new, hydrogel-based, 3D brain-like tissue model to be hosted in the brain compartment of the aforementioned platform. We previously cultured primary mouse microglial cells, cortical neurons and astrocytes independently, once embedded or covered by a millimeter layer of two selected collagen-based hydrogels. We evaluated cell metabolic activity up to 21 days, cell morphology, spatial distribution and synapse formation. Then, we exploited the best performing culturing condition and developed a more complex brain-like tissue model based on the co-culture of cortical neurons and glial cells in physiological conditions. The obtained results indicate that our 3D hydrogel-based brain tissue model is suitable to recapitulate in vitro the key biochemical parameters of brain tissue.

DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation profile of DISP3-expressing cells was altered, as evidenced by delayed expression of neural specific markers and a reduced capacity to undergo neural differentiation.

Purpose SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. Materials and methods To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. Results Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. Conclusion These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.

Sensorineural hearing loss, primed by dysfunction or death of hair cells in the cochlea, is the main cause of severe or profound deafness. Piezoelectric materials work similarly to hair cells, namely, as mechano-electrical transducers. Polyvinylidene fluoride (PVDF) films have demonstrated potential to replace the hair cell function, but the obtained piezoresponse was insufficient to stimulate effectively the auditory neurons. In this study, we reported on piezoelectric nanocomposites based on ultrafine PVDF fibers and barium titanate nanoparticles (BTNPs), as a strategy to improve the PVDF performance for this application. BTNP/PVDF fiber meshes were produced via rotating-disk electrospinning, up to 20/80 weight composition. The BTNP/PVDF fibers showed diameters ranging in 0.160–1.325 μm. Increasing collector velocity to 3000 rpm improved fiber alignment. The piezoelectric β phase of PVDF was well expressed following fabrication and the piezoelectric coefficients increased according to the BTNP weight ratio. The BTNP/PVDF fibers were not cytotoxic towards cochlear epithelial cells. Neural-like cells adhered to the composite fibers and, upon mechanical stimulation, showed enhanced viability. Using BTNP filler for PVDF matrices, in the form of aligned ultrafine fibers, increased the piezoresponse of PVDF transducers and favored neural cell contact. Piezoelectric nanostructured composites might find application in next generation cochlear implants. Keywords: Electrospinning, Barium titanate, Polyvinylidene fluoride, Aligned fibers, Neural cells, Transducer

The foundations of neuroscience are based on the morphological classification of neuronal cells reconstructed in two dimensions where their structure has been correlated to function. Recent advances in microscope technology have facilitated the generation of high resolution three dimensional images of cellular structures containing multiple overlapping data sets. The manual (human expert) analysis of structure and function in these large, multi-dimensional data sets is impractical Computer aided image analysis is used in a variety of fields where digital image data is processed using specialised software for the unbiased interpretation of large data sets. Several applications in medicine use a mix of human and automated techniques for image data analysis, with some of the most challenging data being generated from high resolution imaging of neural cells. Due to the complexity of the data, existing two and three dimensional segmentation and feature extraction techniques may not perform well. This research seeks to develop a robust technique to automatically analyse neural cells following the extraction of functionally relevant data sets of three dimensional images from the central nervous system of animal models.

Johdanto: Kudosteknologia on monitieteellinen tutkimusala, jonka pitkän linjan tavoitteena on tuottaa uusia hoitomuotoja sairauksien hoitoon. Kudosteknologiassa yhdistetään biologiaa insinööritieteisiin ja pyritään laboratorio-oloissa luomaan keinotekoisia kudoksenomaisia viljelmiä, joissa soluja kasvatetaan biomateriaali-tukirakenteessa. Tämä väitöskirja keskittyy ihmisen keskushermoston kudosteknologisiin sovelluksiin. Keskushermosto, kuten muutkin kudokset, koostuu solujen lisäksi soluväliaineesta. Keskushermoston soluväliaine on erittäin pehmeä, mutta elastinen rakenne, joka koostuu nanokokoisista säikeistä. Biomateriaalit, joita käytetään viljelmän tukena voivat jäljitellä kohdekudoksen ominaisuuksia tai toimia mekaanisena tukena solujen kasvulle. Tämän väitöskirjan tavoitteena oli luoda 3D-ympäristö, jota voidaan käyttää solumallin pohjana. Materiaalit ja menetelmät: Tässä väitöskirjatyössä käytettiin ihmisen erittäin monikykyisistä kantasoluista erilaistettuja hermosoluja. 3D-soluviljelyn tukirakenteina käytettiin joko synteettistä PuraMatrix®-hydrogeeliä tai gellan gum -polysakkaridipohjaista hydrogeeliä. Solujen vastetta hydrogeeleihin tutkittiin kolmella erilaisella tutkimusasetelmalla: 1) viljelemällä soluja hydrogeelin alla, jolloin hydrogeeli geeliytettiin suoraan soluviljelmän päälle, 2) viljelemällä soluja esigeeliytetyn hydrogeelipinnan päällä tai 3) viljelemällä soluja sekoitettuna hydrogeeliin. Hydrogeelien lisäksi kolmiulotteisia hermoverkkoja kasvatettiin käyttämällä tukimateriaalina sylinterimäisiä polymeerikomposiitti mikrorakenteita – mikrotorneja. Solujen kasvua tarkasteltiin fluoresoivien leimojen avulla. Tässä väitöskirjatyössä on käytetty sekä yleisiä kaikkiin soluihin sitoutuvia leimoja että vasta-ainetunnistuksen avulla vain kohdeproteiiniinsa sitoutuvia leimoja. Lisäksi hermosoluviljelmien sähköistä aktiivisuutta tutkittiin mikroelektrodihilaan (microelectrode array, MEA) perustuvalla menetelmällä. Tulokset: Eläviin soluihin sitoutuvat fluoresoivat merkkiaineet osoittautuivat hyväksi tutkimusmenetelmäksi. Menetelmän avulla nähtiin, että kahdesta eri viljelmästä peräisin olevat hermosolut pystyivät muodostamaan yhtenäisen hermoverkon, kun ne yhdistettiin samaan viljelmään. Tämän löydöksen soveltaminen jatkossa voi auttaa tutkimuksissa, joissa pyritään selvittämään solusiirrehoidoissa tarvittavaa hermoverkkojen yhteensulautumista. PuraMatrix®¬-hydrogeelin sisällä viljellyt hermosolut kypsyivät muodoltaan monimuotoisiksi sekä niiden sähköinen signalointi oli hermosoluille tyypillistä. Löydösten valossa PuraMatrix® materiaalina vaikuttaa soveltuvan hyvin hermosolujen viljelyalustaksi. Hermosoluille parhaimmat geelipitoisuudet muodostivat hyvin löyhän geelin, minkä johdosta näytteiden valmistus ja käsittely oli haastavaa. Tämä seikka huonontaa PuraMatrix®-hydrogeelin käytettävyyttä hermokudosmallin tukimateriaalina, vaikka muutoin tulokset ovat erittäin lupaavia. Gellan gum -pohjaisia geelejä apuna käyttäen tutkittiin tukimateriaalin jäykkyyden vaikutusta hermosolujen kasvuun ja yritettiin kartoittaa soluviljelyssä käytettävälle tukimateriaalin ihanteellisia ominaisuuksia. Jäykkyydeltään aivoja vastaava materiaali ei kuitenkaan osoittautunut solukokeissa parhaimmaksi. Tämän perusteella todettiin, että jäykkyyden lisäksi monet muut materiaalin ominaisuudet vaikuttavat solujen kasvuun. Parhaat tulokset saatiin gellan gum -hydrogeelillä, johon oli sekoitettu soluväliaineen proteiinia, laminiinia, lisäämään materiaalin bioaktiivisuutta. Hydrogeelien lisäksi sylinterimäiset polymeerikomposiittimikrotornit osoittautuivat erinomaisiksi alustoiksi pienten kolmiulotteisten hermoverkkojen viljelemiseen. Hermoverkkojen muodostuminen mikrotornien ympärille tapahtui toistettavasti ja hermoverkot noudattelivat mikrotornin rakennetta. Tämä työ tukee ajatusta, jonka mukaan kovista materiaaleista valmistettu tukirakenne on hyvä vaihtoehto laboratoriomallin pohjaksi. Johtopäätökset: Väitöskirjatyön pohjalta voidaan todeta, että ihmisen hermosoluille ihanteellisen tukimateriaalin mekaanisten tai kemiallisten ominaisuuksien kartoittaminen ei ole yksioikoista. Monet tutkimukset keskittyvät tuottamaan monikäyttöisiä materiaaleja keskushermoston sovelluksiin. Tämä on haastavaa, sillä erilaisiin koeasetelmiin tarvitaan hyvin erilaisia ominaisuuksia. Tämän työn pohjalta herääkin kysymys siitä, olisiko parempi keskittyä materiaalin yleisen optimoinnin sijasta optimoimaan sen käyttöä soluviljelyalustana valikoiduissa sovelluksissa. Tämä tarkoittaisi materiaalin valitsemista tutkimuskysymyksen mukaan niin, että materiaalin ominaisuudet tukevat parhaalla mahdollisella tavalla tutkimuksen kohteena olevan ilmiön havaitsemista. Background: This thesis focuses on in vitro tissue engineering (TE) applications for the central nervous system (CNS). TE is a multidisciplinary research field that aims at healing human tissues by developing novel engineered biological constructs. Typically, these constructs are composed of cells in a 3D biomaterial scaffold. One promising research platform for neural TE is the use of human stem cell derived neural cells, combined with a 3D scaffold, as a simplified model system for CNS – an in vitro neural model. The commonly used scaffold materials are brain extracellular matrix (ECM)-mimicking hydrogels or rigid microscale 3D matrices that support cell growth. The main aim of this work was to create a 3D neural culturing setup that could serve as a brain tissue model in the future. Materials and Methods: In this thesis, human pluripotent stem cell-derived neural cells were used. The neural cell cultures were visualized with fluorescent labeling, either using cell-accumulating probes or immunocytochemical labeling. Electrical neuronal network functionality was measured using a microelectrode array (MEA) platform. The hydrogels utilized were PuraMatrix®, a synthetic self-assembling hydrogel, and a natural polysaccharide gellan gum hydrogel crosslinked with spermidine (SPD). In this thesis, hydrogels were studied using three culturing methods: 1) Direct gelation of the hydrogel on top of the pre-cultured cell monolayer (2D); 2) Cells cultured on top of the hydrogel surface (2D); 3) Encapsulated cells cultured within the hydrogel (3D). Rigid tubular microtower scaffolds were polymerized using 2 photon-direct laser writing (2PP-DLW) from a polymer-ceramic hybrid material, Ormocomp. Results: Fluorescent probes were found to be suitable for tracking human neural cells for up to four weeks in culture. Co-cultures from two pre-labeled neural populations formed a homogenous neural network in 2D culture, and the electrical neuronal network activity of these co-cultures was similar to that of the control cultures. Both of the studied hydrogel materials were optimized for human neural cell culturing by testing different hydrogel compositions. Interestingly, sparse hydrogels with low PuraMatrix® dilutions were best for neuronal cells, whereas stiff composition of gellan gum hydrogel supported neural networks better than the softer compositions. Neural networks cultured within the PuraMatrix® hydrogel were also shown to have spontaneous electrical activity. Maturation of neuronal activity was slow in the 3D setup; however, on the other hand, morphological inspection showed in vivo mimicking complexity in the cell morphology. Rigid microtower scaffolds were found to be effective for supporting 3D neural network formation around the scaffold. These neural networks were stable for up to four weeks in culture, even though a slight decrease in cell number was seen at the four-week time point. Conclusions: Both of the studied scaffold types, hydrogels and rigid scaffolds, were suitable as a supporting matrix for the formation of 3D neural networks. Large neural networks randomly formed inside hydrogels and were anisotropic, whereas the networks formed around microtowers were smaller, but more easily controlled with the microtower design. All of the studied scaffold materials have potential in in vitro neural applications, but they are applicable to different study questions.

Ihmisen monikykyiset kantasolut ovat kykeneviä jakaantumaan loputtomasti sekä erilaistumalla tuottamaan mitä tahansa ihmiskehon solutyyppiä. Monikykyisiä kantasoluja ovat sekä alkion sisäsolumassasta eristetyt alkion kantasolut, että erilaistuneista soluista geneettisesti muokatut indusoidut monikykyiset kantasolut. Monikykyiset kantasolut voidaan laboratorio-olosuhteissa tehokkaasti ohjata erilaistumaan kaikiksi keskushermoston tärkeimmiksi solutyypeiksi; hermosoluiksi, astrosyyteiksi, ja oligodendrosyyteiksi. Monikykyisistä kantasoluista erilaistettuja keskushermoston soluja voidaan hyödyntää keskushermoston kehityksen ja häiriöiden in vitro mallinnuksessa. Lisäksi monikykyisistä kantasoluista voidaan tuottaa soluja korvaamaan vahingoittunutta kudosta keskushermoston vaurioiden ja sairauksien hoidossa. Keskushermostoon kuuluvan selkäytimen vaurioituminen johtaa tahdonalaisen lihastoiminnan, tuntoaistin, ja autonomisten toimintojen häiriöihin tai katoamiseen. Keskushermoston solujen rajallisen uusiutumiskyvyn vuoksi kantasolusiirteitä on tutkittu paljon yhtenä vaihtoehtona selkäydinvaurion hoitoon. Tällä hetkellä tehokasta hoitoa selkäydinvaurioon ei ole. Tämän väitöskirjatyön tavoitteena oli tutkia eri tekijöiden, kuten solujen geneettisen taustan sekä kemiallisen ja mekaanisen induktion, vaikutusta monikykyisistä kantasoluista erilaistettujen keskushermoston solujen erilaistumiseen ja käyttäytymiseen in vitro kasvatusolosuhteissa. Tuloksia voidaan hyödyntää kehitettäessä kudosteknologisia sovelluksia selkäydinvaurion hoitoon. Ensimmäisessä osatyössä tutkittiin useiden monikykyisien kantasolulinjojen hermosoluerilaistustehokkuutta, ja pyrittiin selvittämään systemaattisia eroja alkion kantasolujen ja indusoitujen monikykyisien kantasolujen välillä. Aiemmat tutkimukset ovat osoittaneet että huolimatta näiden solutyyppien samankaltaisuudesta, niiden alkuperästä johtuvat geneettiset eroavaisuudet saattavat vaikuttaa solujen erilaistumistehokkuuteen. Tulokset osoittivat että solulinjojen välillä on selvästi linjakohtaisia eroja erilaistumistehokkuudessa. Vaihtelua on kuitenkin huomattavasti enemmän yksittäisten solulinjojen kuin eri lähteistä peräisin olevien monikykyisien kantasolutyyppien välillä. Seuraavaksi monikykyisten kantasolujen erilaistusta pyrittiin ohjaamaan kemiallisella induktiolla tarkasti määritellyissä kasvatusolosuhteissa. Erilaisia kasvutekijöitä käytettiin tarkoin ajoitettuna suuntaamaan solujen erilaistumista ensin keskushermoston esiastesoluiksi ja myöhemmin oligodendrosyyttien esiastesoluiksi ja oligodendrosyyteiksi. Menetelmällä saavutettiin oligodendrosyyttien esiastesolujen tehokas erilaistuminen, mutta solujen kypsymistä oligodendrosyyteiksi pitäisi edelleen vakauttaa ja tehostaa. Solujen kohdennettu erilaistus tapahtui ilman eläinperäisiä tekijöitä sisältäviä yhdisteitä, mikä mahdollistaa tällä menetelmällä tuotettujen solujen käytön kliinisissä sovelluksissa. Erilaistusmenetelmästä muokattiin myös kustannustehokkaampi, tutkimustasoinen menetelmä. Soluväliaineen molekyylien vaikutusta monikykyisistä kantasoluista erilaistettujen hermosolujen ja astrosyyttien käyttäytymiseen ja kehitykseen in vitro tutkittiin useilla laminiini-molekyylin isoformeilla. Soluväliaine on solunulkoinen matriisi, joka tukee solujen erilaistumista, kasvua, ja kehitystä sekä rakenteellisesti että kemiallisesti. Tulokset osoittivat että sekä hermosolut että astrosyytit kiinnittyvät ja kasvavat parhaiten pinnoitteilla, jotka sisältävät laminiinin α5-ketjurakenteen. Nämä pinnoitteet myös tehostivat hermosolujen verkostotason toiminnallista kehitystä. Tässä työssä käytetyt pinnoitteet ovat myös kliiniseen käyttöön sopivia, ja niitä voidaan hyödyntää monikykyisistä kantasoluista erilaistettujen keskushermoston solujen tuottoon kudosteknologisia sovelluksia varten. Lopuksi kasvatusolosuhteiden mekaanisen muokkauksen vaikutusta tutkittiin monikykyisistä kantasoluista erilaistetuilla hermosoluilla, astrosyyteillä, ja oligodendrosyyttien esiastesoluilla. Soluja kasvatettiin nanokuitupinnoilla, joiden topografia vastaa selkäytimen solujen suuntautumista in vivo. Nanokuitujen havaittiin vaikuttavan kaikkien solutyyppien kasvuun, ja solujen nähtiin suuntautuvan kasvatusalustan nanokuitujen mukaisesti. Pelkät nanokuidut eivät tukeneet hermosolujen ja astrosyyttien kasvua, mutta kun kuitujen pinta käsiteltiin aiemmin tutkituilla α5-ketjurakenteen sisältävillä laminiini-pinnoitteilla, molempien solutyyppien kasvua voitiin huomattavasti parantaa. Lisäksi osoitettiin, että monikykyisistä kantasoluista erilaistettujen hermosolujen kasvua voidaan ohjata nanokuiduilla myös hydrogeelipohjaisessa 3D-ympäristössä, joka vastaa in vivo kasvuolosuhteita keskushermostossa paremmin kuin 2D pinta. Tämän väitöskirjan tulokset tuovat merkittävää lisätietoa ihmisen monikykyisistä kantasoluista erilaistettujen keskushermoston solujen in vitro –kasvatuksesta. Tulokset ovat hyödynnettävissä kudosteknologisissa sovelluksissa sillä yhdistämällä ihmisperäisiä keskushermoston tärkeimpiä solutyyppejä, kliiniseen käyttöön sopivia kasvatuspinnoitteita, hydrogeelistä koostuva 3D ympäristö ja nanokuitujen ohjaava vaikutus voidaan edistää toiminnallisen solusiirteen kehitystä selkäydinvaurion hoitoon. Human pluripotent stem cells (hPSCs) are capable of self-renewal and differentiation into any cell type found in the human body. hPSCs include embryonic stem cells (ECSs), which are derived from the embryonic inner cell mass, as well as induced pluripotent stem cells (IPSCs), derived from somatic cells with specific transcription factors to induce pluripotency. hPSCs can be efficiently directed to differentiate into the main cell types of the central nervous system (CNS), neurons, astrocytes, and oligodendrocytes in vitro. hPSC-derived neural cells provide an excellent approach to model CNS development and deficits in vitro and furthermore treat injuries and degenerative conditions by replacing damaged tissues. Spinal cord injury (SCI) describes damage to the spinal cord that leads to the loss of muscle function, sensation, or autonomic functions to different extents. CNS cells have limited capacity for regeneration, and cell replacement therapies have been extensively studied for SCI repair, as no efficient treatment for SCI currently exists. This thesis focused on studying various aspects, including genetic background and chemical and mechanical inductions, of hPSC-derived neural cell differentiation and behavior in vitro. The results were aimed to be applicable for developing tissue engineering products for SCI repair. First, the neural differentiation potentials of several hPSC lines were compared in vitro in order to discover the presence of potential systematic differences between hESCs and hIPSCs. Previous studies have demonstrated that despite their considerable resemblance, the different genetic backgrounds of hESCs and hIPSCs may reflect to their differentiation potential. Our results revealed cell line-specific variations in neural differentiation efficiency. However, the variation between individual hESC and hIPSC lines was higher than the variation between cell lines of different origins, indicating the lack of systematic differences between the two hPSC types. Next, chemical induction was studied for the differentiation of hPSC-derived oligodendrocytes. A variety of growth factors and morphogens were used in a temporally defined manner to direct the differentiation through the neural precursor cell stage into oligodendrocyte precursor cells (OPCs), and finally to maturing oligodendrocytes. Although efficient differentiation of OPCs was achieved, the maturation of the cells into oligodendrocytes should be further stabilized and enhanced. The differentiation process was initially performed under xeno-free and defined culture conditions, enabling clinical applications of the produced cells. The protocol was also adapted into a more cost-effective research-grade method. Cellular responses to different extracellular matrix (ECM) molecules in vitro was studied with hPSC-derived neurons and astrocytes. ECM is found at the intercellular space in vivo and it provides both structural support and chemical cues for cell survival, differentiation, migration, and growth. Neurons and astrocytes were cultured on defined ECM substrates using different isoforms or fragments of laminin molecule. hPSC-derived neurons and astrocytes were shown to attach and grow most efficiently on substrates containing laminin α5-chain. These substrates also enhanced the functional development of neuronal networks. The studied substrates were both defined and xeno-free and thus can be used as platforms for the efficient production of clinical-grade neural cells from hPSCs. Finally, the effects of mechanical alterations in the culture conditions were studied with hPSC-derived neurons, astrocytes, and OPCs. The cells were cultured on aligned nanofiber platforms mimicking the aligned cellular orientation in the spinal cord. All cell types were found to orient according to the underlying fiber alignment. However, ECM protein-coating for the nanofibers was required for efficient growth of the neurons and astrocytes, and previously identified laminin α5-chain substrates were successfully applied here. As the 3D cultures resemble in vivo conditions more accurately than traditional 2D culturing surfaces, orientation guidance of hPSC-derived neurons was also demonstrated in 3D culture conditions consisting of nanofiber platform and hydrogel component. In conclusion, the results of this thesis provide new insights for the culturing of hPSC-derived neural cells in vitro. These results can be utilized for tissue engineering applications, as combination of hPSC-derived CNS cells, clinically relevant ECM substrates, 3D environment of the hydrogel and guiding features of the nanofibers would be beneficial for the development of functional cell grafts for SCI repair.