Academic literature on the topic 'Substrate rigidity'

Du cerveau à l’os, la rigidité et la composition de la matrice extracellulaire varient énormément et jouent un rôle dans les réponses cellulaires. La rigidité influe également sur la tension de la membrane plasmique, elle-même régulée par le trafic membranaire. Comment la rigidité et la composition du substrat peuvent réguler l'exocytose, qui à son tour régule la tension de la membrane, reste largement inconnu. Ici, j'ai utilisé l’imagerie pHluorin d’évènements uniques d’exocytose de cellules cultivées sur des substrats de rigidité et de composition contrôlée pour explorer la régulation de VAMP2 et VAMP7. J'ai développé un logiciel informatique pour identifier automatiquement les évènements de fusion, permettant une analyse rapide de données. J'ai contribué à l'étude montrant que l’exocytose VAMP7 est régulée par la kinase src, qui phosphoryle VAMP7 dans son domaine Longin (LD) (Burgo et al. JBC 2013). De plus, j’ai trouvé que la rigidité du substrat stimule l’exocytose, en présence de la laminine, de VAMP7, mais pas VAMP7 sans LD ni VAMP2. VAMP7 et VAMP7 sans LD sont par ailleurs également sensibles aux variations de la tension membranaire induites par chocs osmotiques. Enfin, j'ai identifié que LRRK1 est un partenaire du LD, et contrôle le transport rétrograde de VAMP7.Ces approches m’ont permis de révéler un nouveau mécanisme par lequel la rigidité, agissant sur la signalisation des intégrines, contrôle le transport de VAMP7 via LRRK1 et Rab21 (Wang et al. soumis). Ce mécanisme pourrait avoir un large intérêt potentiel pour comprendre la dynamique de la membrane dans des conditions normales et pathologiques, en particulier le cancer<br>From brain to bones, stiffness and composition of the extracellular matrix vary greatly and play a role in cell responses. Substrate rigidity also impacts plasma membrane tension, which has a close relationship with membrane trafficking. How substrate rigidity and chemistry sensing may regulate exocytosis, which in turn regulates membrane tension, is still largely unknown. Here, I used pHluorin imaging of single vesicle exocytosis in cells cultured on substrates of controlled rigidity and composition to explore the regulation of VAMP2 and VAMP7-mediated exocytosis. I developed a computer software to automatically identify fusion events thus allowing quick analysis of batch data. I contributed to the study showing that VAMP7 exocytosis is regulated by src kinase which phosphorylates VAMP7 in its Longin domain (LD) (Burgo et al. JBC 2013). I further found that VAMP7 but not VAMP7 lacking LD- or VAMP2-mediated secretion was stimulated by substrate stiffness on laminin. VAMP7 and VAMP7 lacking LD were similarly sensitive to osmotic chock-induced membrane tension changes. Finally, i showed that LRRK1, a regulator of egf receptor transport, is a partner of the LD, and controls the retrograde transport of VAMP7. These approaches allowed me to reveal a new mechanism whereby substrate rigidity, by acting on integrin signalling, enhances VAMP7 exocytosis via LRRK1- and Rab21-dependent regulation of its peripheral readily-releasable pool (Wang et al. submitted). This mechanism may have broad potential relevance for plasma membrane dynamics in normal conditions and diseases, particularly cancer

L’ingénierie tissulaire est une stratégie médicale qui repose sur la régénération de tissu par les cellules avec ou sans matériaux. Pour maîtriser cette synthèse, il faut comprendre la cellule comme une part intégrante du tissu. Hormis ses interactions biochimiques avec son support, la cellule interagit également mécaniquement avec son environnement. Elle s’accroche à ce dernier et évalue sa dureté pour adapter sa réponse biologique. Dans cette étude, j’ai développé des modèles numériques pour analyser l’influence de la rigidité du substrat sur le comportement mécanique de la cellule, sur sa structure contractile interne et les efforts qu’elle génère. En d’autres termes, j’ai essayé de comprendre comment la cellule ressent la rigidité de son environnement. De plus, au lieu de me focaliser sur les propriétés mécaniques quantitatives, j’ai cherché à développer un modèle conceptuel simplifié plus proche de la structure cellulaire<br>Tissue engineering is a medical strategy based on utilizing cells and materials to regenerate a new tissue. Yet, it involves intertwined interactions that allow cells to act as integrated parts of an organ. In addition to chemical reactions, the cell interacts mechanically with its environment by sensing its rigidity. Here, we used several computational models to understand how substrate rigidity affects a cell’s structure as it adheres and spreads on it. In other words we tried to understand the way a cell feels how soft or hard it surrounding is, how it affects its internal structure and the forces that transit within it. In addition, instead of focusing on mechanical properties, we developed a simplified, yet coherent conceptual understanding of the cellular structure

"Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering. "

Les cellules s'adaptent en permanence à leur microenvironnement. En particulier, elles modifient leur morphologie, leur croissance, leur division et leur motilité en fonction des propriétés biochimiques et physiques de la matrice extracellulaire (MEC). Elles sont équipées de structures adhésives appelées plaques d’adhérences, permettant aux cellules d'interagir avec les protéines de la MEC via les protéines transmembranaires appelées intégrines et de détecter la nature et la rigidité de la MEC. Le signal est transduit par les protéines des plaques d’adhérences et résulte par exemple en une modification de la tension mécanique induite par l'acto-myosine. Les voies de signalisation en aval peuvent également atteindre le noyau. L'expression des gènes peut alors être modifiée, ce qui peut en retour affecter la composition des plaques d’adhérences et de la MEC pour une réponse cellulaire adaptative. Nous avons émis l'hypothèse qu'en plus des voies de signalisation, un couplage mécanique direct entre les événements se produisant à la périphérie de la cellule et le noyau pourrait participer à la transmission de signaux mécaniques. Bien que les filaments intermédiaires (FIs) aient des propriétés mécaniques extrêmement intéressantes et résistent à des charges de tension élevées, leur implication dans les voies de mécanotransduction est encore mal connue. En utilisant l'astrocyte comme modèle, en raison de sa combinaison spécifique de FIs : vimentine, GFAP, nestine et synémine, nous avons d'abord étudié l'effet de la rigidité du substrat sur la morphologie, la structure et les fonctions du noyau, ainsi que sur l'organisation des FIs autour du noyau. Nous avons ensuite étudié l’impact de l’absence de FI les changements nucléaires observés en réponse à la rigidité du substrat. En utilisant une combinaison de techniques de microfabrication, de méthodes biochimiques et de microscopie, nous avons montré que la rigidité du substrat affecte la forme, le volume du noyau, la structure de la chromatine et le recrutement des facteurs de transcriptions (YAP). Nos résultats suggèrent que les FI forment une structure en forme de cage autour du noyau d'une manière dépendante de la rigidité : un substrat plus rigide induit la formation d’une cage de vimentine et de nestine. Cette interaction avec le noyau pourrait expliquer les modifications nucléaires observées en réponse à la rigidité du substrat. Au total, les résultats obtenus au cours de notre étude permettent de mieux comprendre le rôle des filaments intermédiaires dans les réponses nucléaires aux propriétés mécaniques du substrat<br>Cells continuously adapt to their microenvironment. In particular, they modulate their morphology, growth, division, and motility according to the biochemical and physical properties of the extracellular matrix (ECM). Cells are equipped with adhesive structures called FAs, allowing them to interact with ECM proteins through the core transmembrane proteins called integrins and to sense the nature and the rigidity of the ECM. This information are transduced by FA proteins and lead, for instance, to changes in acto-myosin-mediated mechanical tension. Downstream signalling pathways also reach the nucleus; gene expression is then modified and may, in return, affect the composition of FAs or of the ECM proteins for adaptative cell response. Here, we hypothesized that, in addition to signalling pathways, a direct mechanical coupling between the events occurring at the cell periphery and the nucleus may participate in the transmission of mechanical cues and the regulation of nuclear functions. Although intermediate filaments (IFs) have extremely interesting mechanical properties and resist high tension load, their involvement in mechanotransduction pathways remains elusive. Using astrocyte as a model, due to its specific combination of IFs: vimentin, GFAP, nestin, and synemin, we studied first the effect of substrate rigidity on the nucleus morphology and function, and on the organisation of IFs around the nucleus. Then, we investigated the role of IFs in rigidity-induced nuclear changes. Using a combination of microfabrication techniques, biochemical and microscopy methods, we showed that substrate rigidity affects the nucleus shape, volume, and structure of the chromatin and the recruitment of transcription factor (YAP) and IFs are mediating these changes. Our results suggest that IFs form a cage-like structure around the nucleus in a rigidity-dependent manner: stiffer substrates promote the formation of a cage of vimentin and nestin. In the absence of IFs, the nuclear changes induced by rigidity are different than with IF. The nucleus increases its size in soft substrate, together with an increase in tension measured by YAP localising in the nucleus. The structure of the chromatin is changed. Altogether, the results obtained during our investigation give a better understanding of the role of intermediate filaments in the mechanosensitive nuclear responses

La migration des cellules souches de la pulpe dentaire (DPSCs) est un aspect fondamental de l’ingénierie tissulaire dentaire. L’objectif de cette thèse est de déterminer l’influence de la rigidité du substrat sur la migration des DPSCs. Dans une première partie, nous avons montré que les DPSCs sont capables de survivre et de proliférer sur des substrats de polydiméthylsiloxane (PDMS) avec un module de Young de 1,5 kPa à 2,5 MPa sans se différencier. Nous avons observé que la vitesse moyenne des DPSCs est augmentée sur les substrats de faible rigidité. De plus, la Yes-associated protein (YAP) conserve une localisation nucléaire même sur les PDMS de faible rigidité. Finalement, nous avons montré que sur un substrat comportant deux rigidités différentes, les DPSCs n’adoptent pas de direction de migration préférentielle, contrairement au phénomène de durotaxie classiquement décrit dans la littérature<br>Migration of dental pulp stem cells (DPSCs) is a fundamental aspect of dental tissue engineering. The objective of this thesis is to investigate the influence of substrate stiffness on DPSCs migration. In the first part of this thesis, we showed that DPSCs are able to survive and proliferate on polydimethylsiloxane substrates (PDMS) with a Young's modulus of 1.5 kPa to 2.5 MPa without differentiating themselves. We observed that the average speed of DPSCs is increased on substrates with low stiffness. In addition, the Yes-associated protein (YAP) maintains a nuclear localization even on PDMS with low rigidity. Finally, we have shown that on a substrate with two different stiffnesses, DPSCs do not adopt any preferential migration direction, unlike the process of durotaxis classically described in the literature

L’objectif de ce travail de thèse est de réaliser des substrats et des dispositifs de culture cellulaire pour des applications à grande échelle. En utilisant à la fois des techniques de lithographie conventionnelles et non conventionnelles, nous avons d'abord fabriqué des matrices denses de piliers élastomère avec un gradient de hauteur pour les études de migration cellulaire et nous avons observé un allongement cellulaire remarquable et une migration cellulaire dirigée, tout dépendant du gradient de rigidité. Les micropiliers élastomères pourraient également être organisés en gradient de hauteur oscillant, montrant des comportements cellulaire similaires. Sur la base d'une approche biomimétique, nous avons produit des nanofibres à deux côtés d'une membrane avec des trous traversants pour l’adhésion et la migration tridimensionnelles de cellules. Nos résultats ont montré qu'un tel substrat peut favoriser l'infiltration et la prolifération des cellules dans un environnement 3D. Enfin, nous avons utilisé des réseaux micropiliers de différentes hauteurs en tant que substrat de rigidité contrôlée pour la différenciation des cardiomyocytes à partir de cellules souches pluripotentes l'homme. À l'aide d'un stencil en élastomère, des embryons uniformes pourraient être obtenus et dérivés vers les cellules de ciblage sur le substrat de différentes rigidité, montrant clairement une dépendance de rigidité des substrats<br>The purpose of this work is to develop manufacturable cell culture substrates and devices for large scale applications. By using both conventional and non-conventional lithography techniques, we firstly fabricated dense elastomer pillar arrays with height gradient for cell migration studies and we observed remarkable cell elongation and directed cell migration, all depending on the strength of the stiffness gradient. Elastomer micropillars could also be organized in ripple-like height gradient patterns, showing similar cell behaviors. Based on a biomimetic approach, we produced nanofibers on both side of a membrane with through holes for three-dimensional cell adhesion and migration. Our results showed that such a 3D scaffold can promote the cell infiltration and proliferation. Finally, we used micropillar arrays of different height as stiffness controlled substrate for cardiomyocytes differentiation from human induced pluripotent stem cells (hiPSCs). With the help of an elastomer stencil, uniform embryoids could be obtained and derived to the targeting cells on the substrate of different stiffness, showing a clear stiffness dependence of the substrates

L’objectif de cette thèse est d'étudier l'influence d'hydrogels de faibles rigidité sur l’organisation de la chromatine de cellules épithéliales PtK2 et cancéreuses SW480. Sur des hydrogels mous, la chromatine de PtK2 se structure en hétérochromatine. Les hydrogels très mous conduisent à la nécrose. Sur ces substrats, l'euchromatine, maintenue par inhibition de HDAC, guide la cellule en quiescence. Ces cellules se divisent après transfert sur surfaces rigides. Un processus de dissémination métastatique est développé en cultivant des cellules cancéreuses sur des hydrogels très mous (E20) et des surfaces rigides (verre). Les cellules meurent lors du 1er passage sur E20. Au 2ème passage sur E20, leur survie, motilité et pourcentage en hétérochromatine augmentent. Au 3ème passage, la survie et la motilité progressent cependant le pourcentage en hétérochromatine diminue. Du 1er-2ème passage, les cellules répondent à un processus de dissémination « hétérochromatine­ dépendant » , du 3ème-4ème passage à un processus « euchromatine-dépendant »<br>The aim of this thesis is to investigate the influence of soft hydrogels on the chromatin plasticity of epithelial PtK2 and cancer cells SW480. On soft hydrogels, the chromatin of PtK2 cells is organized in heterochromatin. The very soft hydrogels direct the cell death by necrosis. On these substrates, the euchromatin maintained by inhibition of HDAC guides the cells into quiescence. These cells transferred on stiff substrate enter in mitosis. A process of metastatic dissemination is developed from cancer cells grown on very soft hydrogels (E20) and stiff surfaces (glass). On the 1st seeding on E20, cells die. The 2nd seeding on E20 shows that cell viability, motility and heterochromatin percentage increase. On the 3rd seeding on E20, survival and motility continue to increase while the heterochromatin percentage decrease. From the 1st- 2nd E20 seeding, cells respond to a heterochromatin-dependent process of metastatic dissemination and from the 3rd-4th E20 seeding to an euchromatin-dependent process

La myopathie myofibrillaire est une maladie neuromusculaire à évolution lente caractérisée par de graves troubles musculaires causés par des mutations dans le gène codant pour des protéines du cytosquelette. L'un des gènes affectés en relation avec le développement de la MFM est DES. Des mutations dans le gène de la desmine entraînent des myopathies des muscles squelettiques et cardiaques. Cependant, les évènements qu'elles entraînent et qui sont à l’origine des phénotypes pathologiques cardiaques restent mal connus. Mon objectif est de créer un modèle in vitro de MFM basé sur des cellules souches pluripotentes humaines afin d'étudier le rôle des mutations spécifiques dans la desmine sur le développement et la fonction des cellules cardiaques. Pour atteindre cet objectif, en collaboration avec les docteurs A. Behin, K. Wahbi et la société Phenocell, nous avons généré des iPSC à partir des cellules sanguines périphériques de patients souffrant d'une forme de cardiomyopathie induite par une mutation de la desmine. Les lignées iPSC générées contenant les mutations du gène codant la desmine ont permis d’étudier le rôle d’une mutation dans la spécification et la fonction des cardiomyocytes. La bioénergétique mitochondriale, la structure cellulaire et la fonction contractiles ont été évaluées au niveau cellulaire. En conclusion, il convient de noter que les mutations de la desmine conduisent à une désorganisation des structures des sarcomères dans les cardiomyocytes et à une perturbation de l'expression des protéines mitochondriales. Ce qui conduit à une altération des fonctions de la mitochondrie. Ces données permettent d’améliorer notre compréhension des mécanismes moléculaire qui sous-tendent le développement de la MFM<br>Myofibrillar Myopathy is a slowly progressive neuromuscular disease characterized by severe muscular disorders caused by mutations in the gene encoded cytoskeletal proteins. One of the genes described in connection with the development of MFM is DES. Mutations in the desmin gene lead to skeletal and cardiac muscles myopathies. However, the cardiac pathological consequences caused by them remain poorly understood. My objective is to create an in vitro human stem cell model of MFM to specifically investigate the role of patient-specific mutations in desmin on cardiac lineage development and function. To achieve that objective, in collaboration with Drs. Behin and K. Wahbi and Phenocell, we generate patient-specific iPSC from peripheral blood cells of the patient suffering severel form of desmin-deficient cardiomyopathy. The generated iPSC lines carrying DES gene mutations enable a powerful examination of the role of desmin mutation on cardiomyocyte specification and function. Bioenergetic, structural, and contractile function will be assessed in a single cell. In conclusion, it should be noted that desmin mutations lead to a disorganization of sarcomere structures in cardiomyocytes and to a perturbation of mitochondrial protein expression. This leads to a distortion of functions in the mitochondria. These data facilitate the understanding of the molecular pathway underlying the development of desmin-related myopathy. And the system we have created could also allow us to better evaluate the correlation between the desmin genotype and phenotype in terms of effect on the heart

Dissertação de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.<br>As células estaminais mesenquimais (MSCs) são células estaminais adultas, multipotentes, capazes de se auto renovar e diferenciar em diferentes tipos celulares dentro das linhagens de origem mesenquimal. A colheita de células estaminais mesenquimais é feita a partir de tecidos mesenquimais e também de tecidos extra embrionários. Estes últimos constituem uma boa fonte de MSCs, sendo estas mais naïve e com maior potencial de proliferação do que MSCs de tecidos adultos, características que fazem com que MSCs da matriz do cordão umbilical sejam um tipo celular muito apelativo para experiências de reprogramação. A geração de células estaminais pluripotentes induzidas (iPSCs), nomeadamente a partir de MSCs, tem sido documentada na literatura por diferentes autores, no entanto sempre associada a uma baixa eficiência. É sabido que células estaminais pluripotentes e os seus núcleos têm propriedades elásticas distintas daquelas apresentadas por células diferenciadas e células estaminais adultas (e os seus respectivos núcleos). A partir destas observações colocámos a hipótese de que, através da modulação da rigidez de MSCs, poderíamos aumentar a eficiência do processo de reprogramação usando um vector lentiviral que codifica factores de pluripotência. O núcleo está mecanicamente acoplado a elementos do citoesqueleto através do complexo LINC (ligante do nucleoesqueleto ao citoesqueleto) e desta forma as forças mecânicas vindas da matriz extracelular podem ser transmitidas através do citoesqueleto até ao núcleo. Dependendo da rigidez do substrato, o núcleo está sob maior ou menor tensão, sendo eventualmente possível modular o seu módulo elástico se as células forem plaqueadas em plataformas com diferentes graus de rigidez. Com este trabalho demonstrámos que ao plaquear MSCs em substratos com distintos graus de rigidez, é possível torná-las mais propícias a uma total reprogramação. Para além disto, verificou-se também um aumento na expressão de genes de pluripotência devido apenas ao facto de MSCs serem mantidas em cultura em substratos específicos. Ao analisarmos o estado de compactação da cromatina, bem como a área nuclear tornou-se evidente o efeito que a rigidez dos substratos tem sobre as células. Assim sendo, os rácios do conteúdo eucromático e heterocromático dos núcleos, bem como a área nuclear mostraram ser modulados, o que sugere que os núcleos sofrem mecanomodulação. Foram também observadas diferenças no que ao tamanho das Adesões focais (FAs) e à organização das fibras de actina diz respeito. Tomando em conjunto todos os nossos resultados, verificou-se que é possível melhorar o processo de reprogramação através da modulação da rigidez do substrato, e que o mecanismo por detrás desta melhoria pode estar intimamente relacionado com a mecanomodulação do núcleo. Este progresso na geração de iPSCs humanas, recorrendo a substratos de rigidez definida, dá indícios de que os protocolos habituais de reprogramação celular em plataformas convencionais de cultura de células podem ser substancialmente melhorados plaqueando as células nesses mesmos substratos. Deste modo, aumentado a eficiência e a cinética da geração de células estaminais pluripotentes induzdas, estudos futuros poderão explorar a utilização de vectores de reprogramação não integrativos, considerados mais seguros para possíveis aplicações clínicas.<br>Mesenchymal stem cells (MSCs) are multipotent adult stem cells able to self-renew and differentiate into several cell types within mesenchymal origin, which can be collected from adult mesenchymal tissues, and also from extra-embryonic tissues. The latter constitute a good source of MSCs, being more naïve and having a more proliferative potential than MSCs from adult tissues, features that make umbilical cord matrix MSCs an appealing cell type for the generation of induced pluripotent stem cells (iPSCs). The generation of human iPSCs, namely from human MSCs, has been reported, although with a low efficiency. It is known that pluripotent stem cells and their nuclei possess distinct elastic properties from differentiated and adult stem cells (and respective nuclei). We hypothesize that, by modulating the rigidity of MSCs, it may be possible to enhance the reprogramming efficiency using a lentiviral vector encoding pluripotency factors. The nucleus is mechanically coupled to cytoskeletal elements by the LINC (Linker of Nucleoskeleton to Cytoskeleton) complex, thus mechanical forces from the extracellular matrix can be transmitted through the cytoskeleton to the nucleus. Depending on substrate stiffness, the nucleus is under more or less tension, eventually being possible to modulate its rigidity by culturing the cells on platforms with distinct degrees of stiffness. Here we demonstrated that MSCs plated on substrates with distinct range of rigidity showed different degrees of efficiency to fully reprogram. Moreover, it was shown that maintaining MSCs on specific substrates enhanced the expression of pluripotency genes. The effect of substrate rigidity on the cells was evident when chromatin compaction and nuclear area were analyzed. Thus, nuclear euchromatic and heterochromatic content ratios and area could be modulated, suggesting that nuclei were subjected to mechanomodulation. Differences were also observed in what concerns the size of Focal adhesions (FAs) and the assembling of actin stress fibers. Taken together, our results suggest that it is possible to improve the reprogramming process by modulating the substrate rigidity, and that the mechanism responsible for this improvement could be intimately related with the mechanomodulation of the nuclei. The enhanced generation of human iPSCs cells using substrates with defined stiffness indicates that the current cell reprogramming protocols can be substantially improved by seeding the cells on such substrates. Thus, by improving the efficiency and kinetics of iPSCs generation, future strategies may be further explored using non integrative reprograming delivery strategies, considered safer for putative future clinical applications.

Cell response to substrate rigidity is an emerging field with implications in processes ranging from embryological development to the pathogenesis of disease states such as cancer or fibrosis, in which changes in tissue mechanical properties may inform cellular behavior. It may also serve as a valuable tool in tissue engineering, where materials must be chosen to best influence desired cell phenotype. This thesis describes novel substrates with patterned mechanical properties and their effects on mesenchymal stem cell (MSC) and macrophage behavior. Though substrate rigidity has previously been shown to guide MSC differentiation in two dimensions on unpatterned substrates, differentiation in response to substrates with patterned mechanical properties and in three dimensions has never been demonstrated. Unfortunately, all systems currently used to study these phenomena are limited in their ability to combine spatial patterning of rigidity with cell encapsulation and 3D culture. By altering polymer molecular weight and concentration and using defined mixing and photolithographic patterning techniques, I have developed poly(ethylene glycol) diacrylate hydrogels with tunable rigidity patterned in distinct patterns and gradients. These hydrogels are highly biocompatible and may be crosslinked and patterned under conditions that allow cellular encapsulation and 3D culture. Using these hydrogels, I have shown spatial control over cellular behavior including patterned MSC differentiation in three dimensions in response to substrate rigidity. The potential to drive differentiation in 3D using the mechanical properties of the substrate is particularly exciting, as it bypasses the difficulties of spatially restricting the growth factors currently used to guide progenitor cell differentiation in vitro. In the future, these substrates may be used to engineer tissues with complex architecture in three dimensions.