Induced pluripotent stem cells (iPSCs) have been recognised as a virtually unlimited source of stem cells that can be generated in a patient-specific manner. Due to these cells’ potential to give rise to all differentiated cell types of the human body, they have been widely used to derive differentiated cells for drug screening and disease modelling purposes. iPSCs also garner much interest as they can potentially serve as a source for cell replacement therapy. Towards the realisation of these biomedical applications, this thesis aims to address challenges that are associated with scale-up, safety and biofabrication. Firstly, the manufacture of a high number of human iPSCs (hiPSCs) will require standardised procedures for scale-up and the development of a flexible bioprocessing method, since standard adherent hiPSC culture exhibits limited scalability and is labour-intensive. While the quantity of cells that are required for cell therapy depends largely on the tissue and defect that these replacing cells are meant to correct, an estimate of 1 × 10^9 has been suggested to be sufficient for several indications, including myocardial infarction and islet replacement for diabetes. Here, the development of an integrated, microcarrier-free workflow to transition standard adherent hiPSC culture (6-well plates) to scalable stirred suspension culture in bioreactors (1 L working volume, 2.4 L maximum working volume) is presented. The two-phase bioprocess lasts 14 days and generates hiPSC aggregates measuring 198 ± 58 μm in diameter on the harvesting day, yielding close to 2 × 10^9 cells. hiPSCs can be maintained in stirred suspension for at least 7 weeks with weekly passaging, while exhibiting pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2. These cells retain their ability to differentiate into cells of all the three germ layers in vitro, exemplified by cells positive for AFP, SMA, or TUBB3. Additionally, they maintain a stable karyotype and continue to respond to specification cues, demonstrated by directed differentiation into beating cardiomyocyte-like cells. Therefore, the aim of manufacturing high hiPSC quantities was met using a state-of-the-art scalable suspension bioreactor platform. Secondly, multipotent stem cells such as induced neural stem cells (iNSCs) may represent a safer source of renewable cells compared to pluripotent stem cells. However, pre-conditioning of stem cells prior to transplantation is a delicate issue to ensure not only proper function in the host but also safety. Here, iNSCs which are normally maintained in the presence of factors such as hLIF, CHIR99021, and SB431542 were cultured in basal medium for distinct periods of time. This wash-out procedure results in lower proliferation while maintaining key neural stem cell marker PAX6, suggesting a transient pre-differentiated state. Such pre-treatment may aid transplantation studies to suppress tumourigenesis through transplanted cells, an approach that is being evaluated using a mouse model of experimental focal demyelination and autoimmune encephalomyelitis. Thirdly, biomedical applications of stem cells can benefit from recent advancements in biofabrication, where cells can be arranged in customisable topographical layouts. Employing a 3DDiscovery bioprinter, a bioink consisting of hiPSCs in gelatin-alginate was extruded into disc-shaped moulds or printed in a cross-hatch infill pattern and cross-linked with calcium ions. In both discs and printed patterns, hiPSCs recovered from these bioprints showed viability of around 70% even after 4 days of culture when loaded into gelatin-alginate solution in aggregate form. They maintained pluripotency-associated markers TRA-1-60 and SSEA-4 and continued to proliferate after re-plating. As further proof-of-principle, printed hiPSC 3D constructs were subjected to targeted neuronal differentiation, developing typical neurite outgrowth and resulting in a widespread network of cells throughout and within the topology of the printed matrix. Staining against TUBB3 confirmed neuronal identity of the differentiated cellular progeny. In conclusion, these data demonstrate that hiPSCs not only survive the 3D-printing process but were able to differentiate along the printed topology in cellular networks. / Induzierte pluripotente Stammzellen (iPSZ) stellen eine praktisch unbegrenzte Stammzellquelle dar, welche patientenspezifisch erzeugt werden kann. Da diese Zellen das Potenzial haben, alle differenzierten Zelltypen des menschlichen Körpers hervorzubringen, werden sie für die Herstellung differenzierter Zellen für Arzneimitteltests und für die Krankheitsmodellierung verwendet. Sie erfahren auch großes Interesse, weil sie als Zellquelle in der Zellersatztherapie Anwendung finden könnten. Die vorliegende Dissertation beschäftigt sich mit drei zentralen Herausforderungen, die im Rahmen der biomedizinischen Anwendung von iPSZ auftreten. Die Herstellung einer großen Zahl von humanen iPSZ (hiPSZ) erfordert die Entwicklung standardisierter Verfahren für die Skalierung, welche durch die Entwicklung einer flexiblen Bioprozessmethode realisiert werden kann. Bisher wird die Skalierbarkeit durch eine standardmäßig adhärente Zellkultur und den damit verbundenen hohen Arbeitsaufwand begrenzt. Die Menge an Zellen, die für die Zelltherapie benötigt wird, hängt stark vom Gewebetyp ab, welcher von den ersetzenden Zellen korrigiert werden soll. Berechnungen legen nahe, dass eine Anzahl 1 × 10^9 Zellen für eine Vielzahl von Indikationen ausreicht – einschließlich Myokardinfarkt und Inselzelltransplantation für Diabetes. Im Rahmen dieser Arbeit wurde ein integrierter Arbeitsablauf zur skalierbaren Zellsuspensionskultur von hiPSZ ohne Verwendung von microcarrier entwickelt, um die standardmäßig adhärente Kultur (6-Well-Platten) in Bioreaktoren (1 L Arbeitsvolumen, 2,4 L maximales Arbeitsvolumen) zu überführen. Der zweiphasige Produktionsprozess dauert 14 Tage und erzeugt hiPSZ-Aggregate mit einem finalen Durchmesser von 198 ± 58 μm, der annähernd 2 × 10^9 Zellen beinhaltet. hiPSZ können mindestens 7 Wochen lang in einer gerührten Zellsuspension bei wöchentlichem Passagieren gehalten werden, wobei sie Pluripotenz-assoziierte Marker wie TRA-1-60, TRA-1-81, SSEA-4, OCT4 und SOX2 beibehalten. Die Zellen behalten weiterhin ihre Fähigkeit, sich in vitro in Zellen mit AFP-, SMA- oder TUBB3-Immunoreaktivität und damit in Zellen aller drei Keimblätter zu differenzieren. Darüber hinaus halten sie einen stabilen Karyotyp aufrecht und reagieren auf gezielt eingesetzte externe Differenzierungsstimuli, wie durch eine gezielte Differenzierung in schlagende Kardiomyozyten-ähnliche Zellen demonstriert werden konnte. Somit wurde das Ziel, eine großen Anzahl hiPSCs herzustellen, mit einer hochmodernen, skalierbaren Suspensionsbioreaktorplattform erreicht. Multipotente Stammzellen wie induzierte neurale Stammzellen (iNSZ) gelten verglichen mit iPSZ als sicherere Zellquelle für Ersatztherapien. Die Vorkonditionierung von Stammzellen vor der Transplantation ist jedoch ein heikles Thema, da sowohl die einwandfreie Funktion im Wirtsgewebe als auch Sicherheit gewährleistet werden müssen. Im Rahmen dieser Arbeit wurden iNSZ, die normalerweise im Kulturmedium mit Faktoren wie hLIF, CHIR99021 und SB431542 gehalten werden, für eine definierte Zeitspanne in basalem Medium kultiviert. Die Vorbehandlung führt zu einer geringeren Proliferation, jedoch unter Erhalt der Expression des wichtigen neuralen Stammzellmarkers PAX6, was auf einen transienten vordifferenzierten Zustand hindeutet. Eine solche Vorbehandlung könnte bei zukünftigen Transplantationsstudien angewandt werden, um die Tumorentstehung durch transplantierte Zellen zu unterdrücken. Dieser Ansatz wird in Zukunft mit einem Mausmodell der experimentellen fokalen Demyelinisierung und der autoimmunen Enzephalomyelitis untersucht. Schließlich kann die Zellersatztherapie von den jüngsten Fortschritten in der Biofabrikation profitieren, bei der die Zellen durch das Drucken in anpassbare topographische Profile angeordnet werden können. Mit einem 3DDiscovery Biodrucker wurde eine Biotinte bestehend aus Gelatine-Alginat und hiPSZ in scheibenförmig extrudiert oder in einem Kreuzschraffurmuster gedruckt und mittels Kalziumionen-Zugabe vernetzt. Gedruckte hiPSZ zeigten auch nach 4 Tagen Kultivierung eine Lebensfähigkeit von etwa 70 % und weiterhin das Auftreten der Pluripotenz-assoziierten Marker TRA-1-60 und SSEA-4. Zudem konnten sie sich anschließend mit standardmäßig adhärenter Zellkultur weiter vermehren. Zudem konnte gezeigt werden, dass die gedruckten Konstrukte einer gezielten neuronalen Differenzierung unterzogen werden können, die zu einem typischen Neuritenauswuchs und zu einer weitreichenden interzellulären Vernetzung durch und innerhalb der Topologie der gedruckten Matrix führte. Die Färbung gegen TUBB3 bestätigte die neuronale Identität der differenzierten Zellen. Zusammenfassend zeigen diese Daten, dass bei Verwendung des in dieser Studie erarbeiteten Protokolls hiPSZ nicht nur den 3D-Druckprozess überleben, sondern auch entlang der gedruckten 3D Topologie in Netzwerke Neurone differenzieren können.

Because of the latest developments in nanotechnology and the need to have new functions, a high demand for innovative materials is created. The technological requirements for new applications cannot be fulfilled by most of the well-developed materials like metals, plastics, or ceramics. Therefore, composite materials that can exhibit better properties in contrast to their single counterpart represents a valuable and interesting alternative for the development of new and more performing functional materials. In the past few years, one of the most rapidly developing fields in materials chemistry is research and development of innovative hybrid materials and nanocomposites having exceptional properties. A significant reason for this is that this group of materials closes the gaps between different scientific fields and brings together the ideal properties of the different disciplines into a single system. Conventional materials like polymers or minerals can be mixed with substances of a different kind, like biological molecules and different chemical functional groups to create unique functional materials with the help of a building block method. Inorganic and organic chemistry, physical and biological sciences are integrated in the search for new recipes in a purely interdisciplinary way to generate unique materials. Compounds that are created frequently have interesting new properties for forthcoming functional materials and technological applications. Natural materials frequently function as a model for these systems and various examples of biomimetic methods can be obtained while generating these hybrid materials. The research and development of these materials is driven by the needs of future technologies. The research carried out in this thesis is entirely based on hybrid organic-inorganic materials; hence, it consists of soft organic/bioorganic section that makes it possible to generate multifunctional materials, whereas the hard inorganic section functions as a rigid and stable platform for developing nanocarriers and imaging agents. A key domain in materials chemistry is the creation of smart materials that have the ability to respond to environmental changes or be triggered on demand. These materials have led to the creation of new technologies, like electroactive materials, electrochromic materials, biohybrid materials, sensors and membranes, etc. The required functionality can be provided by the organic or inorganic components, or from both. In this dissertation, the synthesis, methodology, and creation of three unique organic-inorganic hybrid stimuli responsive systems having targeted features for specific applications are examined. The first example is represented by supramolecular microtoroids created by spontaneous self-assembly of amphiphilic molecules and a hydrophilic polymer (chitosan), in the presence of iron (III) chloride. Light irradiation is the stimulus responsible for assembly/disassembly of this new supramolecular entities. The basis of the photo-response of the microtoroids is the photoreaction of the anthracene derivatives. In order to make these materials bio applicable, the microtoroid size was controlled and narrowed down to nanometers, which has led to our second system called metal organic complexes (MOCs). In this system, chitosan was replaced by PNIPAM polymer at optimized concentrations. The reversible thermo-response of MOCs comes from the phase transition ability of PNIPAM. The third hybrid material is the core-shell system consisting of mesoporous organosilica coated with iron oxide nanoparticles, used for cargo delivery and cell imaging. The magnetic-response of the core-shell system results from the strong magnetic properties of iron oxide nanoparticles, while the presence of PMOs increased its biocompatibility. Our research on such organic-inorganic hybrid materials represents a promising development in the field of materials chemistry. Due to the possibility of mixing various properties in a single material, a variety of combinations regarding possible materials and applications have emerged.

Yes / MXenes (viz., transition metal carbides, carbonitrides, and nitrides) have emerged as a new subclass of 2D materials. Due to their outstanding physicochemical and biological properties, MXenes have gained much attention in the biomedical field in recent years, including drug delivery systems, regenerative medicine, and biosensing. Additionally, the incorporation of MXenes into hydrogels has garnered significant interest in biomedical engineering as an electroactive and mechanical nanoreinforcer capable of converting nonconductive scaffolds into excellent conductors of electricity with an impressive effect on mechanical properties for the engineering of electroactive organs and tissues such as cardiac, skeletal muscle, and nerve. However, many questions and problems remain unresolved that need to be answered to usher these 2D materials toward their true destiny. Thus, this review paper aims to provide an overview of the design and applications of MXene-integrated composites for biomedical applications, including cardiac tissue engineering, wound healing, infection therapy, cancer therapy, and biosensors. Moreover, the current challenges and limitations of utilizing MXenes in vivo are highlighted and discussed, followed by its prospects as a guideline toward possible various futuristic biomedical applications. This review article will inspire researchers, who search for properties, opportunities, and challenges of using this 2D nanomaterial in biomedical applications. / Open Access Funding provided by Istituto Italiano di Tecnologia within the CRUI-CARE Agreement.

Rodents are essential models for research on fundamental neurological processing and for testing of therapeutic manipulations including drug efficacy studies. Telemetry acquisition from rodents is important in biomedical research and requires a long-term powering method. A wireless power transfer (WPT) scheme is desirable to power the telemetric devices for rodents. This dissertation investigates a WPT system to deliver power from a stationary source (primary coil) to a moving telemetric device (secondary coil) via magnetic resonant coupling. The continuously changing orientation of the rodent leads to coupling loss/problems between the primary and secondary coils, presenting a major challenge. We designed a novel secondary circuit employing ferrite rods placed at specific locations and orientations within the coil. The simulation and experimental results show a significant increase of power transfer using our ferrite arrangement, with improved coupling at most orientations. The use of a medium-ferrite-angled (4MFA) configuration further improved power transfer. Initially, we designed a piezoelectric-based device to harvest the kinetic energy available from the natural movement of the rodent; however, the harvested power was insufficient to power the telemetric devices for the rodents. After designing our 4MFA device, we designed a novel wireless measurement system (WMS) to collect real-time performance data from the secondary circuit while testing WPT systems. This prevents the measurement errors associated with voltage/current probes or coaxial cables placed directly into the primary magnetic field. The maximum total efficiency of our novel WPT is 14.1% when the orientation of the 4MFA is parallel to the primary electromagnetic field, and a current of 2.0 A (peak-to-peak) is applied to the primary coil. We design a novel controllable WPT system to facilitate the use of multiple secondary circuits (telemetric devices) to operate within a single primary coil. Each telemetric device can tune or detune its resonant frequency independently of the others using its internal control algorithm. / Graduate / 2018-04-26

Atualmente, as nanopartículas superparamagnéticas despertam enorme interesse científico devido sua grande variedade de aplicações em biomedicina, tanto na área de diagnóstico quanto no tratamento de enfermidades. Embora muitos materiais vem sendo estudados, os óxidos de ferro (magnetita e maghemita) apresentam maiores avanços nos estudos para aplicações em medicina. A preferência por óxidos de ferro se deve a baixa toxicidade destas partículas quando comparado as nanopartículas metálicas ou ligas. Entretanto, as nanopartículas destes óxidos possuem baixas magnetizações de saturação que diminuem ainda mais com as sucessivas etapas de recobrimento necessárias para conferir funcionalidade a estas partículas. Desse modo, há uma necessidade atual para o desenvolvimento de nanopartículas superparamagnéticas com elevada magnetização, baixa toxicidade e maior facilidade de funcionalização da sua superfície com biopolímeros e agentes funcionalizantes. Neste trabalho, nanopartículas superparamagnéticas de cobalto metálico e ferrita de cobalto foram sintetizadas e suas propriedades magnéticas foram comparadas com a magnetita. Nanopartículas de cobalto foram escolhidas, pois seu elevado comportamento ferromagnético é menor apenas que o ferro metálico, além do baixo custo de seus reagentes. As nanopartículas magnéticas foram sintetizadas pelos métodos de microemulsão e decomposição térmica (baseado no método poliol) e suas composições química, estrutural, tamanho e distribuição de tamanho foram devidamente determinadas. Além disso, as nanopartículas de cobalto metálico e ferrita de cobalto foram recobertas com ouro utilizando o método de crescimento mediado por semente. Os sistemas microemulsionados utilizados neste trabalho não foram eficientes nem na síntese de nanopartículas estáveis de cobalto metálico nem no seu esperado controle morfológico. Já o método de decomposição térmica resultou em um rigoroso controle de composição química, estrutural e morfológico para as diferentes nanopartículas sintetizadas. O recobrimento com ouro foi efetivo na proteção do núcleo magnético e adicionalmente conferiu estabilidade, baixa toxicidade e bifuncionalidade às nanopartículas magnéticas através do seu fenômeno de ressonância plasmônica de superfície o qual foi preservado na nanoestrutura core@shell. O comportamento superparamagnético das nanopartículas de cobalto metálico recobertas com ouro e sua elevada magnetização de saturação foram expressivamente intensificadas quando comparadas as nanopartículas de magnetita sem recobrimento. Portanto, as nanopartículas sintetizadas neste trabalho apresentam propriedades de superfície e magnéticas otimizadas demonstrando um bom potencial para aplicações em biomedicina como sensores bifuncionais óptico-magnético. / Superparamagnetic nanoparticles have been extensively studied because its wide range of biomedical applications in both diagnostic and therapy areas. Although different materials are currently investigated, superparamagnetic iron oxides nanoparticles (SPION), magnetite and maghemite, are the most extensively studied for applications in medicine. The lower toxicity profile of the SPION becomes the most attractive than metal or alloys nanoparticles. Nevertheless, iron oxides nanoparticles have low saturation magnetization, which further decreases due to successive coats to provide their functionality, leading the actual demand to develop superparamagnetic nanoparticles with high magnetization, low toxicity and easy surface functionalization with biocompatible agents. In this work, superparamagnetic nanoparticles of metallic cobalt and cobalt ferrite were synthesized and their magnetic properties were compared with the magnetite SPION. Cobalt nanoparticles were chosen because present high ferromagnetic behavior among chemical elements, second only to iron, besides their low cost. The magnetic nanoparticles were synthesized by both microemulsion and thermal decomposition (based on the polyol process) methods and their chemical composition, structure, size and size distribution were properly characterized. In addition, the ferrite and metallic cobalt nanoparticles were coated with gold by using the seed-mediated growth method. The used microemulsion systems were not efficient enough to synthesize stable metallic nanoparticles and to promote the expected morphological control even to ferrites. Instead, the thermal decomposition processes resulted in rigorous control of chemical compositional, structure and morphology in all different prepared samples. Au-coating process was effective to protect the magnetic nuclei also giving additional stability, low toxicity and a bifunctionality to the magnetic nanoparticle since their surface plasmon resonance phenomenon was preserved in the core@shell nanostructure. The superparamagnetic behavior of the Au-coated cobalt nanoparticle was preserved and their saturation magnetization was significantly increased compared with the naked magnetite SPION. In conclusion, the synthesized nanoparticles present enhanced magnetic and surface properties showing good potential to be used in biomedical application as bifunctional optical-magnetic sensor.

The biodegradability and when functionalised biocompatibility of aliphatic polycarbonates (APCs) makes them an attractive class of materials for biomedical applications such as tissue engineering scaffolds and drug-delivery carriers. One route to accessing a wide-range of well-defined and functional APCs is the controlled ring-opening polymerisation (ROP) of cyclic carbonates. In turn, these would ideally be prepared by the direct coupling of CO2 with diols to give water as the only by-product. In this way, the combination of CO2 and sugar-derived diols draws upon two natural renewable building blocks for the construction of polycarbonates that are anticipated to show good biocompatibility properties. Chapter 2 develops a simple and mild alternative to the traditional use of phosgene derivatives for the synthesis of six-membered cyclic carbonates from 1,3-diols and CO2. DFT calculations highlighted the need to lower both the CO2-insertion and ring-closing kinetic barriers to cyclic carbonate formation. Organic superbase, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) enabled the formation of carbonate species at 1 atm CO2 pressure whereas, the introduction of a leaving group strategy lowered the cyclisation barrier. Mechanistic considerations suggested a kinetic preference for ring- closing via a nucleophilic addition-elimination pathway rather than a SN2-like intramolecular cyclisation. Chapter 3 applies the procedure with CO2 to the preparation of a novel monomer from natural sugar, ᴅ-mannose. ROP was carried out via an organocatalytic approach and a preference for head-tail linkages in the polycarbonate backbone indicated by NMR spectroscopy and supported by DFT calculations. Chapter 4 utilises CO2 to invert the natural stereochemistry of sugars and create a thymidine-based monomer. The thermodynamic parameters of the ROP with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) catalyst are determined and the properties of the polycarbonates investigated to include preliminary cell attachment studies. Finally, chapter 5 details the synthesis of cyclic carbonates from 2- deoxy-ᴅ-ribose and the investigation into the different ROP behaviour of the α- and β- anomers. The ability to tune the polymer properties through copolymerisation with trimethylene carbonate (TMC) is also discussed.

Patient-specific induced pluripotent stem cells (iPSCs) emerged as a promising cell source for disease modeling and drug screening as well as a virtually unlimited source for restorative therapy. The thesis deals with three major topics to help realizing biomedical applications with neural stem cells. To enable the generation of transgene-free iPSCs, alternatives to retroviral reprogramming were developed. Hence, the adaptation and evaluation of reprogramming using excisable lentiviral constructs, Sendai virus (SeV) and synthetic mRNA-based methods was assessed in the first part of this thesis. hiPSCs exhibit the pluripotency markers OCT4, SSEA-4, TRA1-60 which were confirmed by immunofluorescence and flow cytometry. Besides, the potential to differentiate in cell types of all three germ layers was detected, confirming pluripotent identity of proliferating colonies resulting from various reprogramming strategies. However, major differences such as high efficiency with SeV in contrast to a relatively low efficiency with mRNA in regard to passage number and the phenotype of starting fibroblasts were observed. Furthermore, a prolonged clone- and passage-dependent residual presence of viral RNA genes was identified in SeV-iPSCs for up to 23 passages using RT-PCR underlining the importance of careful monitoring of clone selection. In contrast, viral-free reprogramming by synthetic mRNA represents a fully non-integrative approach but requires further refinement to be efficiently applicable to all fibroblasts. The second part of this thesis deals with the establishment of a rapid monolayer approach to differentiate neural progenitor cells from iPSCs. To achieve this, a two-step protocol was developed allowing first the formation of a stable, primitive NPC line within 7 days which was expanded for 2-3 passages. In a second step, a subsequent adaptation to conditions yielding neural rosette-like NPCs followed. Both neural lines were demonstrated to be expandable, cryopreservable and negative for the pluripotency marker OCT4. Furthermore, a neural precursor identity including SOX1, SOX2, PAX6, Nestin was confirmed by immunofluorescence and quantitative RT-PCR. Moreover, the differentiation resulted in TUJ1-positive neurons and GFAP-positive astrocytes. Nonetheless, the outcome of glial differentiation from primitive NSCs remained low, whereas FGF/EGF-NPCs were efficiently differentiated into GFAP-positive astrocytes which were implicated in a cellular model of the blood brain barrier. The third and major objective of this study was to generate human early neural progenitor cells from fetal brain tissue with a wide neural differentiation capacity. Therefore, a defined medium composition including small molecules and growth factors capable of modulation of crucial signaling pathways orchestrating early human development such as SHH and FGF was assessed. Indeed, specific culture conditions containing TGFβ inhibitor SB431542, SHH agonist Purmorphamine, GSK3β inhibitor CHIR99021 and basic FGF, but no EGF enabled robust formation of early neuroepithelial progenitor (eNEP) colonies displaying a homogeneous morphology and a high proliferation rate. Moreover, primary eNEPs exhibit a relatively high clonogenicity of more than 23 % and can be monoclonally expanded for more than 45 passages carrying a normal karyotype. Characterization by immunofluorescence, flow cytometry and quantitative RT-PCR revealed a distinct NPC profile including SOX1, PAX6, Nestin and SOX2 and Prominin. Furthermore, primary eNEPs show NOTCH and HES5 activation in combination with non-polarized morphology, indicative of an early neuroepithelial identity. Microarray analysis unraveled SOX11, BRN2 and other HES-genes as characteristic upregulated genes. Interestingly, eNEPs were detected to display ventral midbrain/hindbrain regional identity. The validation of yielded cell types upon differentiation indicates a strong neurogenic potential with more than 90 % of TUJ1-positive neurons. Moreover, astrocytes marked by GFAP and putative myelin structures indicating oligodendrocytes were identified. Electrophysiological recordings revealed functionally active neurons and immunofluorescence indicate GABAergic, glutamatergic, dopaminergic and serotonergic subtypes. Additionally, putative physiological synapse formation was observed by the presence of Synapsin and PSD-95 as well as by ultrastructural examination. Notably, rare neurons stained positive for the peripheral neuronal marker Peripherin suggesting the potential of eNEPS to give rise to cells of neural tube and neural crest origin. By the application of specific differentiation protocols an increase of TH-positive neurons or neural crest-derivatives such as putative A- and C-sensory neurons and mesenchymal cells was identified. Taken together, primary eNEPs might help to elucidate mechanisms of early human neurodevelopment and will serve as a novel source for cell replacement and further biomedical applications. / Patientenspezifische induziert pluripotente Zellen (iPSZ) haben sich als eine vielversprechende Möglichkeit erwiesen Zellen zu gewinnen, die für Krankheitsmodellierung, Arzneimitteltests und Zellersatztherapie in Frage kommen. In dieser Arbeit wurden drei wichtige Fragestellungen adressiert, die für potenzielle biomedizinische Anwendungen von neuralen Stammzellen von großem Interesse sind. Um die Generierung von transgenfreien iPSZ zu ermöglichen, wurden Alternativen zur retroviralen Reprogrammierung entwickelt. Im ersten Teil dieser Arbeit wurden Reprogrammierungsmethoden, die auf deletierbaren, lentiviralen Konstrukten oder nichtintegrativen Verfahren wie Sendaivirus (SeV)-Transduktion und Transfektion synthetischer mRNA basieren, adaptiert und evaluiert. Die daraus resultierenden iPSZ exprimieren die Pluripotenzmarker OCT4, SSEA-4 und TRA1-60. Weiterhin wurde das Potenzial in Zelltypen aller drei Keimblätter zu differenzieren nachgewiesen. Dadurch konnte die pluripotente Identität der proliferativen Kolonien bestätigt werden. Beim Vergleich der angewandten Methoden fielen, bezüglich der generierten iPSZ-Linien, sowohl qualitative als auch quantitative Unterschiede auf. Bei der Verwendung von SeV-Partikeln wurde eine hohe Reprogrammierungseffizienz festgestellt. Bei der Transfektion von mRNAs hingegen war die Reprogrammierungseffizienz deutlich niedriger. Diese war darüber hinaus abhängig von der Passage und dem Genotyp der Ausgangsfibroblasten. Des Weiteren konnte eine klon- und passagenabhängige Präsenz viraler Gene in SeV-iPSZ bis zu 23 Passagen lang beobachtet werden, während bei der mRNA-Transfektion keine Spuren der genetischen Manipulation zurückblieben. Dies verdeutlicht die Bedeutung einer sorgfältigen Qualitätskontrolle bei der Klonselektion im Falle der SeV-iPSZ. Im Gegensatz dazu stellt die Reprogrammierung durch Transfektion synthetischer mRNAs eine völlig nicht-integrative Strategie dar, erfordert allerdings weitere Verfeinerung um das Verfahren effizient und vor allem für alle Fibroblastenpräparationen anwendbar zu machen. Der zweite Teil der Arbeit behandelt die Etablierung eines schnellen, adhärenten Protokolls, um neurale Vorläuferpopulation aus iPSZ zu differenzieren. Um dies zu erreichen, wurde ein zweiphasiges Protokoll entwickelt, welches zunächst die Generierung einer primitiven neuralen Vorläuferzellpopulation innerhalb von 7 Tagen erlaubt. In einem zweiten Schritt erfolgte die Adaptierung an Kulturbedingungen, die eine neurale, rosettenähnliche Zellpopulation induzieren. Beide neuralen Zellpopulationen konnten weiter expandiert und eingefroren werden und waren negativ für den Pluripotenz-assoziierten Transkriptionsfaktor OCT4. Darüber hinaus konnte die neurale Vorläuferidentität mittels positiver Expression von SOX1, SOX2, PAX6 und Nestin bestätigt werden. Eine weitere Differenzierung dieser Zellen resultierte in TUJ1-positiven Neuronen und GFAP-positiven Astrozyten, die die Verwendung der Zellpopulation beispielsweise in einem zellulären Modell der Blut-Hirn-Schranke erlaubten. Das Hauptprojekt dieser Dissertation war es, frühe humane neurale Vorläuferzellen aus fetalem Hirngewebe zu isolieren und in Kultur zu stabilisieren. Diese Population sollte eine breite Differenzierungskapazität aufweisen. Zu diesem Zweck wurde eine chemisch definierte Medienzusammensetzung gewählt, die zusätzlich pharmakologisch wirksame Verbindungen und Wachstumsfaktoren beinhaltet. Hierdurch konnten Signaltransduktionswege wie zum Beispiel der Sonic-Hedgehog- (SHH) oder FGF-Signalweg, die bei der frühen neuralen Entwicklung eine bedeutende Rolle spielen, moduliert werden. In der Tat ermöglichten spezifische Kultivierungsbedingungen, die den TGFβ-Inhibitor SB431542, den SHH-Agonisten Purmorphamin, den GSK3β-Inhibitor CHIR99021 und basisches FGF, jedoch kein EGF enthielten, die robuste Bildung einer früheren neuroepithelialen Vorläuferpopulation (eNEP). Die so stabilisierten Kolonien wiesen eine homogene Morphologie und eine hohe Proliferationsrate auf. Außerdem zeigten sie eine hohe Klonogenitätsrate von 23%, die es ermöglichte monoklonale Zelllinien zu isolieren und für mehr als 45 Passagen zu expandieren. Dabei blieb ein normaler Karyotyp erhalten. Die Zellen zeigten ein eindeutiges neurales Profil, gekennzeichnet durch SOX1, PAX6, Nestin, SOX2 und Prominin-Expression. Weiterhin wiesen eNEPs NOTCH und HES5-Aktivierung in Kombination mit nicht-polarisierter Morphologie auf, was auf eine frühe neuropitheliale Identität hinweist. Eine Microarray-Analyse demonstrierte weiterhin SOX11, BRN2 und einige HES-Gene als charakteristisch hochregulierte Gene. Interessanterweise zeigen eNEPs eine regionale Identität, die auf eine Mittelhirn/Hinterhirn-Regionalisierung hinweist. Die Validierung ungerichtet ausdifferenzierter Zelltypen offenbarte mit einem Kulturanteil von 90% TUJ1-positiven Neuronen ein stark neurogenes Potenzial. Zusätzlich konnten GFAPpositive Astrozyten sowie mögliche Myelinstrukturen, die auf Oligodendrozyten hinweisen, nachgewiesen werden. Elektrophysiologische Aufzeichnungen deuten auf funktionell aktive Neurone hin und Immunofluoreszenzfärbungen zeigten GABAerge, glutamaterge, dopaminerge und serotonerge neuronale Subtypen. Außerdem wurden mittels Immunfluoreszenzanalyse Synapsin- und PSD-95- positive synaptische Strukturen nachgewiesen. Ultrastrukturelle Analysen mittels Transmissionselektronenmikroskopie bestätigten das Ergebnis. Hervorzuheben ist, dass einige Neurone positiv für den peripheren Neuronenmarker Peripherin gefärbt wurden, was darauf hinweist, dass eNEPs das Potenzial besitzen, in Zellen der Neuralleiste zu differenzieren. Durch die Verwendung von spezifischen Differenzierungsprotokollen konnte das Vorkommen TH-positiver und auch möglicher A- und C-sensorischer Fasern, sowie mesenchymaler Zellen nachgewiesen werden. Zusammenfassend lässt sich sagen, dass primäre eNEPs dazu beitragen könnten, die frühe humane Gehirnentwicklung zu verstehen. Darüber hinaus stellen eNEPs eine potentielle zelluläre Quelle für Zellersatztherapien und weitere biomedizinische Anwendungen dar.

This thesis presents strategies for constructing multi-functional biomaterials based on hyaluronan (HA) derivatives for various biomedical applications, such as drug delivery, tissue regeneration, and imaging biomaterials. The aim of this study is to improve the functionalities of HA biomaterials as well as simplify the preparation procedures.  Native HA polymer contains D-glucuronic acid residue with a carboxyl group per disaccharide unit that can be easily modified by carbodiimide-mediated amidation reaction. Therefore, we have designed a series of orthogonal groups (hydrazide, carbazate, aldehyde, and thiol) that can be linked to HA under mild conditions using the carbodiimide chemistry. Multiple functionalities can be introduced to the obtained HA derivatives via chemoselective “click”-type transformations.   The modified HA derivatives were used for the preparation of either nanogel particles (NPs) or bulk hydrogels. Due to “click” character of the reactions used, structural HA transformations were performed with high fidelity on different scales including molecular (polymers), nanometer (NPs), and a visible scale (bulk hydrogels). By linking pyrene or camptothecin to hydrophilic HA backbone, amphiphilic polymers were obtained and utilized as drug delivery carriers or prodrugs, respectively. Subsequently, physically loaded drug (doxorubicin) could be released upon degradation of HA carriers, while the chemically linked camptothecin was released intact by a thiol-triggered cleavage reaction. Bisphosphonated HA (HA-BP) polymers were prepared to induce hydrogel scaffold bio-mineralization for bone regeneration application. Moreover, we could recruit strong binding capacity of bisphosphonate (BP) groups to calcium ions for the formation of physically crosslinked HA-BP gel upon simple mixing of the polymer and calcium phosphate nanoparticle components. This gel was more stable in vivo compared to hydrazone crosslinked HA gels. Furthermore, the hydrogel composed of fluorine-19 (19F) linked HA polymer was successfully observed by both 1H and 19F MR imaging.         In conclusion, the presented herein study describes new approaches for building up multi-functional biomaterials from the HA-based blocks. The utilization of carbodiimide and click chemistries along with the enzymatic degradation of HA allowed simple and efficient interconversion between HA macromolecules, nanoparticles and macroscopic hydrogels. These HA-based biomaterials show high potential for use in the fields of drug delivery, bone regeneration, and imaging techniques.

Dans les biotechnologies, les particules magnétiques sont de plus en plus utilisées dans diverses applications, de thérapies ou de diagnostics : « Drug delivery », traitements de cancers, IRM, etc. Inscrit dans le démarrage d'un nouvel axe de recherche du laboratoire SPINTEC, le travail de thèse a mis en œuvre des particules élaborées par une approche de type «top-down». Les matériaux utilisés sont antiferromagnétiques synthétiques (SAF). L'intérêt des nanoparticules magnétiques dans les domaines biomédicaux a été présenté en introduction. L'étude réalisée à SPINTEC a tout d'abord consisté à caractériser et modéliser différents matériaux SAF, et développer les procédés technologiques permettant la fabrication des particules. Ensuite le travail a été consacré à l'observation et la modélisation du comportement des particules en suspension dans un champ magnétique appliqué. Un modèle des phénomènes d'autopolarisation et d'agglomération des particules SAF a permis de déterminer une susceptibilité seuil en dessous de laquelle les particules restent dispersées en solution, évitant les phénomènes d'agglomération. / In the biotechnologies, the magnetic particles are more and more used in different applications, therapy or diagnostics such as: drug-delivery, cancer treatments, MRI …This thesis, which marks the beginnings of a new research area at SPINTEC laboratory, implemented particles made by an approach top-down. The used materials are synthetic antiferromagnetic (SAF). The interest for the magnetic nanoparticles was shown in the introduction. The study made at SPINTEC was first of all the characterization and modeling of different SAF materials, and the development of the technological processes for making the particles. Then the work was devoted to observation and modeling of the behavior of the particles in suspension in the applied magnetic field. A model of self-bias and agglomeration of SAF particles allowed to determine a susceptibility threshold below which the particles remain dispersed in solution, avoiding the agglomeration phenomena.

Depuis plusieurs décennies, les polyesters aliphatiques (polycaprolactone (PCL), polylactide (PLA), polyglycolide (PGA)) et leurs copolymères ont été retenus pour des applications médicales grâce à leur biodégradabilité et leur biocompatibilité. Parmi leurs applications médicales, on s’intéresse ici à la délivrance des médicaments par des copolymères amphiphiles et à l’ingénierie tissulaire. Les polyesters aliphatiques souffrent cependant d’une hydrophobie importante et de l’absence de groupes fonctionnels. Pour pallier ces problèmes, plusieurs stratégies demodifications chimiques ont été proposées dans la littérature parmi lesquelles on cite : l’hydrolyse, la modification par plasma, la post-polymérisation alcyne azoture et la modification photochimique thiol-yne. Ces modifications servent à introduire des polymères hydrophiles (ex. le polyéthylène glycol) ou des groupes fonctionnels qui peuvent améliorer la biocompatibilité de polyesters. Dans ce manuscrit, on s’intéresse à la modification de la PCL et du PLA par voie photochimique thiol-yne qui présente l’avantage d’être rapide, versatile, applicable en solution comme en surface et de ne pas nécessiter l’utilisation d’un catalyseur métallique qui peut être nocif pour les applications médicales. Dans une première partie, la modification de la PCL a été faite en solution et des copolymères amphiphiles PCL-g-PEG ont été synthétisés. La stratégie de greffage « grafting to » en deux étapes a été choisie en partant de polymères commerciaux. Une optimisation des conditions de modification par voie anionique de PCL, suivi d’une photoaddition thiol-yne, nous a permis d’obtenir des copolymères avec des balances hydrophiles/hydrophobes contrôlées. L’impact de l’hydrophilie des copolymères sur la formation de nanoobjets, leurs concentrations d’aggrégation critique et leurs tailles a été étudié. L’encapsulation de curcumine comme agent anticancéreux et la cytotoxicité des nanovecteurs envers des cellules cancéreuses ont été vérifiées. Dans un second temps, ces copolymères ont été décorés par un peptide de ciblage et un peptide clivable enzymatiquement en vue de leur utilisation dans des traitements anticancéreux. L’effet biologique de ces copolymères encapsulant des principes actifs est vérifié in vitro sur des cellules cibles exprimant plus ou moins d’intégrines ou de métalloprotéases. Dans une seconde partie, des fibres PLA ont été modifiées en surface par des nanoparticules inorganiques afin de générer des hybrides covalents d’intérêts pour des applications en ingénierie tissulaire. De manière analogue aux modifications en solution, ces hybrides ont été obtenus en deux étapes par modification par voie anionique de nanofibres de PLA, suivi par un greffage covalent de nanoparticules d’oxyde de fer en suivant une stratégie photochimique thiol-yne. / For decades, aliphatic polyesters (polycaprolactone (PCL), polylactide (PLA), polyglycolide (PGA)) and their copolymers have been selected for medical applications because of their biodegradability and their biocompatibility. Among their medical applications, we are interested in drug delivery system based on amphiphilic copolymers and tissue engineering. However, aliphatic polyesters suffer from significant hydrophobicity and the absence of functional groups. To overcome these drawbacks, several strategies ofchemical modifications have been reported in literature among which we present: hydrolysis, plasma modification, post polymerization modification by copper catalyzed azide alkyne cycloaddition and thiol-yne post polymerization modification. These modifications have been used to introduce hydrophilic polymers (eg. polyethylene glycol) or functional groups on the polyester chains that can enhance the biodegradability of polyesters. In this manuscript, we are interested in modifying PCL and PLA chains by thiol-yne photochemical route. This method is rapid, versatile, applicable in solution as well as on surface and it does not require the use of a metallic catalyst which can be harmful for medical applications. First, PCL modification was done in solution and amphiphilic copolymers PCL-g-PEG were synthesized. The strategy “grafting to” in two steps has been selected starting from commercial polymers. Conditions optimization of anionic activation, followed by thiol-yne photoaddition, allowed us to obtain copolymers with controlled ratios hydrophilic/hydrophobic. The impact of copolymers hydrophilicity on nanoobjets formulation, critical micelle concentration and sizes was studied. Curcumin encapsulation as an anticancer agent and nanocarriers cytotoxicity towards cancer cells were verified. In addition, these copolymers were then decorated with a targeting peptide and an enzymatically cleavable peptide in the aim of using them in cancer treatment. The biological effect of anticancer loaded copolymer was verified in vitro on target cells expressing more or less integrins or metalloproteases. Second, PLA fibers were modified with inorganic nanoparticles and generate covalent hybrids for purposes in tissue engineering of neuronal cells. Analogously to the solution modification, these hybrids were obtained in two steps by anionic activation of PLA fibers, followed by covalent grafting of iron oxide nanoparticles according to a thiol-yne photochemical strategy.