In recent years, the degradation and subsequent loss of tissues is an issue that has affected people worldwide. Although there are treatments addressing the degradation of tissues, such treatments involve complicated and expensive procedures, where full tissue regeneration is not achieved. For these reasons, in recent years, tissue engineering has developed cutting-edge biomaterials capable of inducing effective tissue regeneration both under cellular or acellular conditions. Peptide hydrogels are versatile biomaterials composed of the basic components of life amino acids, which act as building blocks to form hierarchical structures, which subsequently go on to form well-defined scaffolds. Biomaterials have been widely used for the culture of mammalian cells, tissue engineering, regenerative medicine, drug delivery, etc. This is thanks to their capability of providing a three-dimensional architecture to cells, which mimics the natural architecture of the extracellular matrix (ECM). Peptide- based hydrogels can be easily functionalised with active biological cues, which can direct the cellular response. It has been shown that the ionic-complementary FEFEFKFK hydrogel, succeeded to support the culture of mammalian cells such as bovine chondrocytes. In this work, we used the same FEFEFKFK hydrogel to investigate the capability of this hydrogel to support the three-dimensional culture of both human osteoblasts (hOBs), and human mesenchymal stem cells (hMSCs) for bone regeneration applications. To achieve this goal, hOBs were cultured within both FEFEFKFK (non-functionalised) and RGD-FEFEFKFK (functionalised) gels. Then the suitability of the FEFEFKFK gels to induce cellular proliferation, synthesis of bone ECM and mineralisation was explored. In addition, taking advantage of the inherent plasticity of hMSCs, we also investigated the capability of the FEFEFKFK gel to foster the osteogenic differentiation of hMSCs, and subsequently to induce bone mineralisation in 3-D under osteogenic stimulation. Based on the results obtained in this work, the FEFEFKFK gel arises as a promising biomaterial for both bone and dental tissue regeneration applications.

Células-tronco humanas derivadas da polpa dental (hDPSCs) e células-tronco humanas derivadas de tecido adiposo (AhSCs) são células multipotentes capazes de diferenciação osteogênica in vitro e in vivo, e promissoras fontes de células para a engenharia de tecido ósseo, dada a sua facilidade de expansão, isolamento e diferenciação. É de grande interesse compreender qual é o melhor tipo celular para diferenciação osteogênica, assim, o objetivo deste estudo foi comparar o potencial de diferenciação osteogênica in vitro e in vivo entre hDPSCs e hASCs. Foram isoladas e estabelecidas seis populações de células-tronco de hDPSCs (entre 7-12 anos) e seis da hASC (de indivíduos com idade entre 30-49 anos). Após a indução in vitro, a diferenciação osteogênica foi comprovado através das colorações de fosfatase alcalina (9 dias) e vermelho de alizarina (14 e 21 dias). A quantificação da mineralização da matriz após 21 dias de diferenciação osteogênica revelou 2,24 mais ossificação das hDPSCs em relação às hASCs. Para realizar o experimento in vivo, foram triados seis biomateriais para verificar qual melhor biomaterial para o nosso modelo, defeito crítico em calvária de Ratos Wistar não imunossuprimidos, com três amostras de hDPSCs. Após 45 dias, CellCeram(TM) exibiu a melhor neoformação óssea in vivo, e foi selecionado para comparar os potenciais osteogênicos in vivo entre hDPSCs e hASCs. Células (10e6) foram associadas a discos de 4,5 mm CellCeram(TM), grupo controle foi realizado através do transplante do biomaterial livre de células. Neoformação óssea foi mensurada 45 dias após a cirurgia através da coloração histológica de hematoxilina / eosina. A formação óssea total foi quantificada através da análise de imagens de todas as ilhas de ossificação. A associação entre hDPSCs e CellCeram(TM) promoveu 7,24 vezes mais neoformação óssea quando comparado com a associação entre esse mesmo material e hASCs (p <0,0001). A utilização de células-tronco adultas para regeneração óssea é uma ótima abordagem para uso terapêutico, e calcular ou predizer o potencial osteogênico das células utilizadas é extremamente importante e necessário para futura aplicação em novas estratégias de bioengenharia de tecido ósseo / Human dental pulp stem cells (hDPSCs) and human adipose-derived stem cells (AhSCs) are multipotent cells capable of undergoing osteogenesis in vitro and in vivo, and promising cell-source populations for bone tissue engineering given their easiness of isolation, expansion and differentiation. It is of great interest to understand which is the best cell type for osteogenic differentiation, thus the aim of this study is to compare the in vitro and the in vivo osteogenic differentiation potentials between DPSCs and ASCs. We isolated six stem cell populations from DPSCs (aged 7-12 years) and six from ASCs (from subjects aged 30-49 years) and cell culture was established. After in vitro induction the populations were able to undergo osteogenic differentiation, as evidenced by alkaline phosphatase (9 days) and alizarin red S (14 and 21 days) stainings. Quantification of matrix mineralization after 21 days of osteogenic differentiation revealed an enhancement of 2.24-fold increase between hDPSCs and hASCs differentiation. To perform the in vivo experiment, we promoted a screening of six scaffolds to find out which would be best scaffold to our model, a calvarial critical-sized defect in Wistar non-immunosuppressed rats, with three different culture samples of hDPSCs. After 45 days, CellCeram(TM) displayed the best in vivo bone neoformation, and was used to compare the in vivo osteogenic potentials between hDPSCs and hASCs. Cells (10e6) were associated to 4.5 mm CellCeram(TM) discs, and control groups were performed transplanting the biomaterial free of cells. Bone healing was measured through histological hematoxylin/eosin staining 45 days after surgery. Newly formed bone was also evaluated by total bone island surface quantification through image analysis. The association between hDPSCs and CellCeram(TM) induced a mean of 7.24 times more bone formation when compared to the association between this same material and hASCs (p<0.0001). The use of adult stem cells for bone regeneration is a robust therapeutic option, and calculate or predicts the osteogenic potential of the cell used are extremely important and necessary to future application, and translation to new strategies in bone tissue engineering

There is an unmet need to develop and test treatments for rare lysosomal disease (LD). Most LDs are present in childhood and do not currently have approved therapies. Rare diseases individually are uncommon but taken together account for a population prevalence of 3.5-5.9% worldwide. Due to their rarity, it often takes significant time and effort to diagnose rare diseases. New diagnostic tools, especially for early detection, will offer an advantage in avoiding this diagnostic odyssey. This dissertation is focused on investigating novel diagnostic and treatment methods in vitro for two neurodegenerative LDs: Gaucher disease (GD) and mucopolysaccharidosis IIIB (MPS IIIB). Mutations in NAGLU and GBA1, the genes that encode for lysosomal hydrolases required for degradation of heparan sulfate and glucocerebrosides, lead to the observed pathogenesis in MPS IIIB and GD, respectively. Since many LDs, including MPS IIIB and some forms of GD, are neurodegenerative, cell and gene-based therapeutic strategies are of significant interest. Therapeutics that offer some symptom mitigation in other LDs, such as enzyme replacement or substrate reduction therapies, do not offer appreciable disease mitigation in MPS IIIB or neurodegenerative GD. Here, a novel compound heterozygous mutation, NAGLUY140C/R297X, that results in approximately 50% residual NAGLU protein and 0.6% NAGLU enzyme activity is reported in NAGLU. Furthermore, a RFLP and site-directed mutagenesis strategy was developed to identify the presence of the relatively common p.R297X mutation in patient cell samples, in addition to two other novel molecular assays for the detection of the p.E153K mutation in NAGLU and p.N370S mutation in GBA1. MPS IIIB and GD human skin fibroblasts were reprogrammed to iPSCs using non-integrating Sendai viral vectors with a reprogramming efficiency of 0.2% and 0.3%, respectively. Resulting iPS cell lines were confirmed as being pluripotent through a barrage of analyses for markers of pluripotency and differentiation. Intriguingly, early passage MPS IIIB iPSCs were found to exhibit increased cell death and spontaneous differentiation to embryoid body-like structures, which was hypothesized to be caused by fibroblast growth factor 2 (FGF2) sequestration or degradation due to inherent heparan sulfate dysregulation. Supplemental FGF2 (100 ng/mL) was found to significantly increase confluency of MPS IIIB iPSCs after 48 hours (n = 5, p ≤ 0.05) and persisting to 96 hrs (n = 5, p ≤ 0.05), thus providing evidence for an important role of FGF2-heparan sulfate interactions in the maintenance of stem cell pluripotency. These findings highlight the importance of considering inherent disease pathology when developing disease models. Three genome editing strategies, CRISPR-Cas9, base and prime editing, are addressed throughout this dissertation. Genome editing outcomes in NAGLU and GBA1, as well as a control gene, HPRT1, are reported in HEK293 cells, human skin fibroblasts, and induced pluripotent stem cells (iPSCs). Although CRISPR-HDR failed to yield mutation correction, base editing of the common p.N370S (c.1226 A>G) in GD skin fibroblasts using with 42% efficiency is reported. Base editing of HPRT1 in HEK293 cells with an overall editing efficiency of 6 ± 0.5% (n = 3), but interestingly, when base editing at the centered nucleotide was analyzed, the editing efficiency increases to 27 ± 4.3% (n = 3). These findings align with other reports of a centered nucleotide preference for base editors and will help direct genome editing strategies in the future. This dissertation describes the first genome editing in NAGLU, and the first base editing in GBA1, and underscores the importance of optimizing genome editing strategies when targeting disease-causing mutations in patient-derived cells. The findings reported here will direct future genome editing strategies for developing cell and gene-based therapies for MPS IIIB and GD. / Graduate / 2021-12-15

abstract: Cellular assays are the backbone of biological studies - be it for tissue modeling, drug discovery, therapeutics, or diagnostics. Two-dimensional (2D) cell culture has been deployed for several decades to garner physiologically relevant information and predict data before the cost-intensive animal testing. Although 2D techniques have been valuable for cellular assays, they have a colossal limitation - they do not adequately consider the natural three-dimensional (3D) microenvironment of the cells. As a result, they sometimes provide misleading statistics. Therefore, it is important to develop a 3D model that predicts cellular behaviors and their interaction with neighboring cells and extracellular matrix (ECM) in a more realistic manner. In recent biomedical research, various platforms have been modeled to generate 3D prototypes of tissues, spheroids, in vitro that could allow the study of cellular responses resembling in vivo environments, such as matrices, scaffolds, and devices. But most of these platforms have drawbacks such as lack of spheroid size control, low yield, or high cost associated with them. On the other hand, Amikagel is a low cost, high-fidelity platform that can facilitate the convenient generation of tumor and stem cell spheroids. Furthermore, Amikabeads are aminoglycoside-derived hydrogel microbeads derived from the same monomers as Amikagel. They are a versatile platform with several chemical groups that can be exploited for encapsulating the spheroids and investigating the delivery of bioactive compounds to the cells. This thesis is focused on engineering novel 3D tumor and stem cell models generated on Amikagel and encapsulated in Amikabeads for proximal delivery of bioactive compounds and applications in regenerative medicine. / Dissertation/Thesis / Z-stacks of confocal images of spheroids encapsulated in Amikabeads (compilations of sections) / Masters Thesis Bioengineering 2020

Despite a comprehensive mapping of the Parkinson's disease (PD)-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2) in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs) and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22648号 / 医科博第111号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23084号 / 医博第4711号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 森本 尚樹, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Vápenaté fosforečnany jsou nejčastěji využívané keramické materiály v medicíně pro regeneraci kostní tkáně. Vápenaté fosforečnany jsou biokompatibilní, bioaktivní a mezi sebou se odlišují především rozpustností a související degradací v organismu, proto jsou nejčastěji využívány k regeneraci malých defektů nebo jako vrstvy na kovových implantátech. U již zmíněné rozpustnosti materiálu po implantaci, hraje důležitou roli poměr zastoupení jednotlivých vápenatých fosforečnanů, od kterého se pak odvíjí rychlost degradace materiálu v organismu. Tato práce se zabývá vlivem tvarovacích metod na mikrostrukturu a zejména fázové složení vápenatých fosforečnanů. Výchozím materiálem pro pozorování změn ve fázovém složení byl komerční hydroxyapatit, který byl upraven kalcinací při 800 °C po dobu jedné hodiny. Při kalcinaci došlo k vytvoření dvoufázové směsi, obsahující hydroxyapatit a -fosforečnan vápenatý. Tato dvoufázová směs byla poté využita pro přípravu suspenze s plněním 15 obj.%, a také k přímé přípravě polotovarů s různou výslednou mikrostrukturou a fázovým složením. S cílem zjistit vliv procesu byly v této práci porovnány vzorky připravené pomocí metod: freeze-casting, izostatické lisování za studena, uniaxiální lisování a suspenzní odlévání. U polotovarů a slinutých vzorků byla provedena charakterizace mikrostruktury a analýza fázového složení. V práci se potvrdil vliv tvarovací metody na oba stanovené parametry – mikrostrukturu a fázové složení. Nově pak bylo zjištěno, že se zvyšující se hodnotou porozity a velikostí pórů se zvyšuje i zastoupení hydroxyapatitu ve vzorcích. Tento trend byl pozorován u vzorků vykazujících unimodální i bimodální zastoupení velikosti pórů.