No description available.

No description available.

No description available.

No description available.

No description available.

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to uniquely serve a structural function providing support and strength to cells within tissues, is increasingly being recognized to have pleiotropic effects in neurogenesis and regeneration processes such as neocortex folding, stem cell niche maintenance, peripheral nerve regeneration, axonal growth, and many more. ECM mediates these processes via cell-ECM interactions which provide the cells with a wealth of signals including biophysical and mechanical cues in a spatiotemporal manner. Owing to the importance of the surrounding microenvironment, modern neural tissue engineering strategies have focused on the development of engineered biomaterials capable of finely instructing the neuronal response according to their physicochemical characteristics. Neurons and neural stem cells are in fact sensitive to their mechanical and topographical environment, and cell–substrate binding contributes to this sensitivity by activating specific signaling pathways for basic cell function. In addition, the advances in nanotechnology have opened the possibility of introducing decorative nano-motifs that interact with cells at the molecular level. Successful strategies in tissue engineering are driven by not only advances in the synthesis of highly instructive biomaterials but also greatly depend on the right selection of cell sources. As a matter of fact, advances in neural tissue engineering have been strongly hampered by the poor availability of cell sources, considering that primary neurons are the only type of cells that do not proliferate. The discovery of induced pluripotent stem cells (iPSCs) has addressed many of the cell-related limitations in neural tissue engineering, offering the possibility to consistently produce a wide range of neural cell lines. Advances in cell biology have led to the development of iPSCs-derived brain spheroid, which surely represent the most promising tools for several neural tissue engineering applications ranging from in vitro modelling of neurodegenerative diseases (i.e., Parkinson's, Huntington's and Alzheimer's), biomaterials testing and drug screening platforms. The overarching goal of my doctoral work was to engineer biomaterials with instructive physicochemical properties to elicit beneficial cellular responses that are suitable for different neural tissue engineering applications such as nerve regeneration and 3D in vitro modelling. In the first study (Chapter 2), I evaluated the compounded effects of surface stiffness and micro-topography on dorsal root ganglion and human bone-marrow mesenchymal stem cells behavior. To this end, arrays of parallel microchannels of different geometries were introduced on the surface of chitosan films by electrophoretic replica deposition. In addition, a novel chemical crosslinking with citric acid was performed to both enhance the long-term stability of the chitosan films and fine-tune the surface stiffness for the investigation of its role in cell behavior. In the second study (Chapter 3), I developed a novel nanocomposite consisting of a collagen hydrogel decorated with glycine-derived carbon nanodots (Gly-CNDs). After a comprehensive physicochemical characterization of the resulting nanocomposite, I evaluated the effects exerted on neuronal differentiation and electrophysiological maturation of mouse iPSCs-derived brain spheroid. In the third study (Chapter 4), I optimized an alignable collagen-based hydrogel characterized by anisotropically oriented fibers with potential applications in both peripheral and central nervous system repair. I established a protocol that encompasses the introduction in the collagen solution of biodegradable laminin-functionalized magnetic microbeads and the time-controlled application of an external magnetic field. The regenerative potential of the hydrogel was unveiled using mouse iPSCs-derived neural stem cells.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24081号 / 理博第4848号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 川口 真也, 准教授 船山 典子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Autism spectrum disorder (ASD) encompasses a group of heterogeneous disorders that affect approximately 1% of children worldwide. ASD is characterized by two core symptoms, the first being deficits in social communication and interaction, and the second being restrictive and repetitive behaviours. Although environmental and genetic factors are known to contribute to the development of ASD, the etiology remains unknown. Genetic sequencing studies have implicated over 1000 genes with risk variants that are ASD-associated. Recent sequencing studies have highlighted that SCN2A, a gene that encodes the Voltage-Gated Sodium Channel Type II Alpha Subunit habours a large proportion of genetic risk variants for ASD. An emphasis was put on this gene because many of the top genes regulate transcription and cytoskeletal dynamics and not sodium flux aiding in regulating neuron excitability. Initial investigations of complete loss of Scn2a in mice led to perinatal lethality where heterozygous loss exhibited many behavioural phenotypes associated with ASD. Through our collaboration with Dr. Stephen Scherer (Hospital for Sick Children, Toronto) we identified two de novo truncating point variants in SCN2A. In our study, we focused on using human iPSC-derived neurons for disease modelling. We found these two variants caused a reduction in synapses suggesting that neuronal communication may be altered. Furthermore, electrophysiological characterization of the neurons harbouring the differing SCN2A variants showcased that loss-of-function (LoF) variants can produce differential phenotypes based on their location. Beyond the initial ion channel characterization, we wanted to probe whether cellular pathways were altered directly or indirectly by atypical neuronal activity. Proteomics of neurons expressing the more severe variant, p.R607*, found differentially expressed proteins (DEP)s that were upregulated and downregulated. Moreover, these DEPs were enriched and clustered into cellular pathways that were altered, with one of these clusters representing mitochondrial function. We functionally validated these findings in the same neurons and found corroboration between the molecular and cellular data of impaired mitochondria. Lastly, we used Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing to generate an isogenic model to validate our findings of the less severe p.G1744* variant. Together, this will aid in the discovery of new variant categorizations and targeted treatments for rescues of atypical neural connectivity or pathways that are altered downstream. / Thesis / Doctor of Philosophy (PhD)

<p>Human pluripotent stem cells (hPSCs) exist as a heterogeneous population within a dynamic niche, which governs their ability to self-renew and differentiate. Evidence modeled after mouse embryonic stem cells (mESCs) reveals the existence of a developmentally primitive, or homogeneous, state through chemically defined culture methods that is modulated by NANOG, a core pluripotent regulator. However, the differentiation potential and transcription factor control of the homogeneous state in human pluripotent stem cells remains elusive. Previous work suggests that bFGF/ACTIVIN extrinsic regulation provides the heterogeneous nature of hiPSCs with ability to differentiate into several multilineage lineage progenitors. Here, we illustrate that altering the extrinsic environment of hiPSCs with LIF and inhibitors of GSK3b and MAPK/ERK1/2 pathways (LIF/2i), rewires the intrinsic pluripotent regulation of OCT4 and NANOG, which ultimately prevents the <em>in vitro</em> hematopoietic differentiation potential. Upon conversion of hiPSCs to a primitive state of pluripotency with LIF/2i, this study reveals that prolonged culture of hiPSCs with LIF/2i erases the hematopoietic differentiation potential through retained expression of the POU domain pluripotent transcription factor, OCT4. Interestingly, shRNA mediated knockdown of <em>OCT4</em> recovers the restricted differentiation potential in LIF/2i cultured hiPSCs, while knockdown of <em>NANOG</em>, does not. This study identifies a distorted differentiation potential of hPSCs cultured in mouse ESC conditions, despite comparable gene expression profiles and signaling pathway dependence. In efforts to simplify culture methods of human pluripotent stem cells, we identify that alteration of the extrinsic environment highlights explicit differences between human and mouse intrinsic pluripotent regulation, which ultimately controls differentiation efficiency.</p> / Master of Science (MSc)

The cannabinoid system exerts functional regulation of neural stem cell (NSC) selfrenewal, proliferation, and differentiation during both homeostatic and pathologic conditions. Recent evidence suggests that cannabinoid signaling is neuroprotective against reduction in NSC proliferation and neurogenesis caused by a multitude of conditions including injury due to HIV-1 associated neurotoxic proteins, neuroinflammation, and stroke. Yet not all effects of cannabinoids or cannabinoid-like compounds on neurogenesis can be attributed to signaling through either of the classical cannabinoid receptors CB1 or CB2. The recently de-orphaned GPR55 is targeted by numerous cannabinoid compounds suggesting GPR55 may be causing these aberrant effects. Activation of GPR55 has shown immune-modulatory effects outside the central nervous system (CNS) and anti-inflammatory actions on microglia, the resident immune cells within the CNS. New evidence has confirmed that both human and murine NSCs express functional levels of GPR55 yet the effects that GPR55 activation has on adult neurogenesis or NSC responses to inflammation has not been elucidated. In the present study we sought to determine the role GPR55 signaling has on NSC proliferation and neurogenesis as well as possible neuroprotective mechanisms within the NSC pool in response to inflammatory insult. Activation of GPR55 increased human NSC proliferation in vitro as assessed by BrdU incorporation and flow cytometry. Neuronal differentiation was also upregulated by signaling through GPR55 under homeostatic conditions in both human and murine NSC samples. Expression of NSC differentiation markers (nestin, sox2, GFAP, S100b, DCX, bIII-tubulin) in vitro was determined by immunohistochemistry, qPCR, and flow cytometry. In vivo, C57BL/6 and GPR55-/- mice were administered the GPR55 agonist O-1602 (4 μg/kg/day) directly into the left hippocampus via stainless steel cannula connected to an osmotic mini-pump for a continuous 14 days. O-1602 treatment increased hippocampal NSC proliferation, survival, and immature neuron formation as compared to vehicle treated animals. These results were determined to be dependent on GPR55 activation as GPR55-/- animals did not show any response to agonist treatment. Interestingly, GPR55-/- mice displayed significantly reduced rates of hippocampal NSC proliferation and neuroblast formation as compared to C57BL/6 animals. Chronic production of inflammatory mediators, such as IL-1b seen in neuroinflammation, to NSCs is known to reduce proliferation rates and attenuate neurogenesis both in vitro and in vivo. Addition of GPR55 agonists to IL-1b (10 ng/mL) treated human and murine NSC samples in vitro protected against reductions in neuron formation as assessed by immunohistochemistry and flow cytometry. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55-/- mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602 (4μg/kg/day) in C57BL/6 mice but not in GPR55-/-mice. Neuroprotection by O-1602 treatment was not found to be microglia dependent as microglia activation was not altered by agonist administration. Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55-/- animals manifesting in a prolonged inflammatory response (IL-1b, IL-6, TNFa) after chronic, systemic inflammation as compared to C57BL/6 animals. Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult. / Biomedical Sciences