The main function of mesenchymal stem cells in the body is to facilitate the restoration and regeneration of damaged tissues. They are known for the ability to differentiate into tissue originating from the mesoderm, which among others includes connective tissues. Due to this feature are MSCs being intensively examined. Different directions of differentiation can be induced by treatment of specific polypeptides, so called growth factors. In the field of tissue engineering are growth factors used to induce and accelerate the healing processes. They may be incorporated into the nanofiber carrier which is inserted into the site of injury. Cells in this area would thus be stimulated by surrounding 3D microenvironment. At the same time the scaffold provides a supply of growth factors which are able to affect metabolism, motility and differentiation of present cells. In order to induce osteogenic differentiation of human MSCs the following bioactive substances were used: TGF-β, bFGF, HGF, IGF-1, VEGF and the BMP-2 and the organic acid taurine. During 21 days lasting experiments, were these molecules added to the medium in various combinations and in the case of taurine also at two different concentrations. Cells were cultured on plastic. The best effect on cellular metabolism of MSCs, evaluated by MTS...

Chaque année 610 000 cancers sont diagnostiqués dans le monde induits par une infection à papillomavirus humains à haut-risque (HPV-HR). Bien que les carcinomes des voies aéro-digestives supérieures (VADS) soient principalement associés à une forte consommation de tabac et d’alcool, 20 à 25% sont causés par une infection à HPV, particulièrement l’HPV de type 16. Les patients HPV positifs présentent un meilleur survi global, pourtant ils sont diagnostiqués avec plus de métastases à distance que les patients HPV négatifs. Au travers d’une étude sur des lignées cellulaires dérivées des VADS, nous avons montré que toutes les lignées cellulaires HPV+ présentaient une intégration du génome d’HPV au sein du génome cellulaire, avec des profils d’intégration différents. Les lignées pouvant être utilisées comme modèles caractéristiques des tumeurs HPV+ et HPV- sont respectivement les lignées UPCI:SCC090 et FaDu. La première par ses capacités migratoire et proliférative et la seconde par sa faible agressivité et une mutation du gène cellulaire p53. Dans une étude portant sur une série rétrospective de cancers de l’oropharynx éligible à une résection chirurgicale, 6 cancers sur 40 soit 15% présentaient une infection à HPV16 active (expression de l’ARNm E6*I). Nous avons étudié les marqueurs de TEM dans ces cancers oropharyngés en fonction du statut HPV. Nous avons retrouvé une perte plus importante du marqueur épithélial cadhérine-E au sein du groupe HPV+, associée à une moins bonne survie globale.Au total, nous montrons que le statut HPV et les marqueurs de TEM semblent être deux facteurs indépendants, qui peuvent se combiner pour définir des niveaux pronostiques différents. / Each year, 610,000 cancers are diagnosed worldwide attributed to high risk human papillomavirus (HR-HPV) infection. Although head and neck squamous cell carcinoma (HNSCC) is mainly associated with tobacco and/or alcohol consumption, 20 to 25% are caused by HPV infection, particularly HPV type 16. Although patients with HPV+ tumors present a better overall survival, they are diagnosed with more lymph node metastasis than HPV-negative patients.Through a study of HNSCC derived cell lines, we showed that all HPV-positives cell lines harbored HPV genome integration through host genome, with different integration profiles. Cell lines identified as good HPV+ and HPV- tumors models are UPCI:SCC090 and FaDu respectively. The first one by its migratory and proliferative properties, the second through its poor aggressiveness and mutation of p53 cellular gene.In a study on a retrospective series of oropharyngeal carcinomas with surgical resection, 6 out of 40 cancers shown HPV16 active infection (expressing E6*I mRNA). We studied epithelial-to-mesenchymal transition (EMT) markers on this oropharyngeal cancers, according to HPV status. We found a larger loss of epithelial marker E-cadherin in HPV+ group and loss of this marker is associated with a worse overall survival.We showed that HPV and EMT status seem to be two independent factors that could combine differently to define different prognostic levels.

Abstract Breast cancer, the most common cancer in women in Finland; its prognosis varies from very good to poor. During the last two decades, mesenchymal stromal cells, carcinoma-associated fibroblasts and normal fibroblasts of the breast have been investigated in the context of breast carcinomas because of their presence in the tumor microenvironment. It has been shown that the properties of the non-malignant tumor compartment possess prognostic value. The effects that these three stromal cell types have on cancer progression have been studied, but their exact mechanisms remain still largely unknown. This experimental work was conducted in order to investigate whether the three cell types of mesenchymal origin influence breast cancer cell proliferation in vitro and tumor growth in vivo. Functional and structural differences between the stromal cell types were investigated using multiple methods. A total of 19 primary human bone marrow derived mesenchymal stromal cell lines, and six paired primary fibroblast and carcinoma-associated fibroblast lines of the breast were used in the study. Co-cultures of labeled stromal cells and breast cancer cell lines MDA-MB-231, M-4A4 and NM-2C5 were performed and the proliferation properties of each cell line were assessed. An orthotopic murine breast cancer model was established by injecting NM-2C5 cancer cells in the mammary fat pads of athymic mice either alone or with the three stromal cell types, and tumor growth and histology were analysed. Mesenchymal stromal cells increased the proliferation of breast cancer cell lines NM-2C5 and MDA-MB-231, and carcinoma-associated fibroblasts increased the proliferation of NM-2C5 cells in vitro. The effect was due to both soluble factors and direct cell-cell contact. In the in vivo experiments, the mesenchymal stromal cells inhibited and the fibroblasts enhanced the growth of breast cancer tumors. Histological analysis of the tumors revealed differences in the invasiveness, necrosis and amount of connective tissue. Differences in the expression of CD105 and CD54 were observed between tumors with mesenchymal stromal cells or fibroblasts. Carcinoma-associated fibroblasts differed from mesenchymal stromal cells in their expressions of CD105 and CD54. The fibroblast subtypes differed at the gene expression level in immunological, developmental and extracellular matrix related pathways. / Tiivistelmä Rintasyöpä on Suomessa naisten yleisin syöpä, ja sen ennuste vaihtelee erittäin hyvästä huonoon. Viime vuosikymmenten aikana mesenkymaalisia stroomasoluja, rinnan kasvainsidekudossoluja ja tavallisia sidekudossoluja on tutkittu rintasyövän yhteydessä johtuen kyseisten solujen läsnäolosta syövän mikroympäristössä. Syöpäkudoksen hyvänlaatuisen solukon ominaisuuksilla on osoitettu olevan ennusteellista arvoa, ja kolmen edellä mainitun strooman solutyypin vaikutuksia rintasyövän etenemiseen on tutkittu, mutta tarkat vaikutusmekanismit ovat vielä laajalti tuntemattomat. Tutkimuksen tarkoituksena oli tutkia edellä mainittujen solutyyppien vaikutusta rintasyöpäsolujen lisääntymiseen soluviljelmässä ja syöpäkasvaimen kasvuun koe-eläinmallissa. Lisäksi strooman solujen rakenteellisia ja toiminnallisia eroavaisuuksia tutkittiin molekyylibiologisilla menetelmillä. Tutkimuksessa käytettiin 19:ää luuytimen mesenkymaalista stroomasolulinjaa sekä kuutta rinnan kasvainsidekudossolu–sidekudossolu paria. Leimattuja strooman soluja viljeltiin yhteisviljelmissä rintasyöpäsolulinjojen MDA-MB-231, M-4A4 ja NM-2C5 kanssa, ja kunkin solutyypin lisääntymistä mitattiin. Ortotooppisessa rintasyövän hiirimallissa immuunipuutteisen hiiren rinnan ihonalaisrasvaan injisoitiin NM-2C5-rintasyöpäsoluja yksinään ja yhdessä strooman solujen kanssa, ja kasvainten kasvua ja histologiaa analysoitiin. Mesenkymaaliset stroomasolut kiihdyttivät NM-2C5- ja MDA-MB-231-rintasyöpälinjojen ja kasvainsidekudossolut NM-2C5-solujen lisääntymistä soluviljelmässä. Vaikutuksen aiheuttivat sekä liukoiset tekijät että suora solujen välinen vuorovaikutus. Eläinmallissa mesenkymaaliset stroomasolut hillitsivät mutta sidekudossolut lisäsivät rintasyöpäkasvaimen kasvua. Histologisissa analyyseissä paljastui eroavaisuuksia tuumorien paikallisessa invaasiossa, kudoskuolion määrässä ja sidekudoksen määrässä. Mesenkymaalisia stroomasoluja ja kasvainsidekudossoluja sisältävien kasvainten välillä esiintyi eroja CD105- ja CD54-pinta-antigeenien määrässä. Kasvainsidekudossolut erosivat pintarakenteiltaan mesenkymaalisista stroomasoluista CD105:n ja CD54:n ilmentämisessä. Sidekudossolut ja kasvainsidekudossolut erosivat toisistaan geenien ilmentämisen tasolla immunologisten, kehityksellisten ja soluväliaineeseen liittyvien geenipolkujen osalta.

La sclérodermie systémique (ScS) est une maladie rare et incurable, caractérisée par une fibrose cutanée et la production d’auto-anticorps (maladie auto-immune). Le pronostic vital est engagé dans les formes diffuses et rapidement progressives de la maladie, responsables de fibrose pulmonaire. Par leurs propriétés immunomodulatrices et anti-fibrotiques, les cellules souches mésenchymateuses (CSM) constituent une approche prometteuse pour traiter la ScS. Ce travail a pour objectif d’évaluer le potentiel thérapeutique des CSM dans un modèle préclinique de forme diffuse de la maladie (d-ScS).Patients et Méthodes : Nous avons évalué l’effet de différentes modalités d’injections des CSM par voie intraveineuse dans le modèle murin de ScS-HOCl. Ce modèle, basé sur l’injection d’acide hypochloreux (HOCl), induit un phénotype proche des formes d-ScS. Nous avons notamment comparé un traitement préventif et curatif, des approches syngénique, allogénique et xénogénique, et l’utilisation de CSM d’origine adipocytaire (ASC) à celle de CSM de moelle osseuse (CSM-MO).Résultats : Le modèle ScS-HOCl est caractérisé par l’installation d’un phénotype de d-ScS, avec fibrose cutanée, pulmonaire et production d’anticorps anti-topoisomérase 1. Nous montrons l’effet bénéfique d’une injection préventive ou curative de CSM syngéniques, réduisant la fibrose cutanée et pulmonaire. Cet effet thérapeutique passe par une diminution de la réponse immune réduisant l’inflammation tissulaire et la production d’auto-anticorps, ainsi que par l’induction du remodelage tissulaire via l’activation de métalloprotéases, et l’augmentation des défenses anti-oxydantes. Un bénéfice similaire est obtenu lors d’approches allogénique et xénogénique. Les ASC présentent des capacités immunosuppressives et de remodelage supérieures aux CSM-MO.Discussion et conclusion : Nos travaux démontrent l’effet anti-fibrotique des CSM dans un modèle préclinique pertinent de ScS, mimant les formes diffuses et rapidement progressives de la maladie, pour lesquelles les besoins thérapeutiques sont importants. Le mode d’action pléiotropique des CSM, combinant propriétés anti-inflammatoires, pro-remodelage et anti-oxydantes, est prometteur en vue des applications cliniques à venir dans cette maladie.Mots-clés : sclérodermie systémique, cellules souches mésenchymateuses, HOCl / Systemic sclerosis (SSc) is a rare intractable disease characterized by skin fibrosis and autoimmunity. Diffuse and rapidly progressive SSc (d-SSc) is associated with life-threatening involvements such as lung fibrosis, where there still is an unmet medical need. Displaying immunomodulatory and antifibrotic properties, mesenchymal stem cells (MSC) are an attractive cure for SSc. In this study, we aim at evaluating the therapeutic potential of MSC in a preclinical model of d-ScS.Patients and Methods: We evaluated the effects of MSC infusion in the murine model of ScS-HOCl, based on repeated injections of hypochlorite (HOCl). We compared several approaches using MSC in a preventive and curative approach, in syngeneic, allogeneic and xenogeneic settings, and using MSC isolated from adipose tissue (ASC) or bone marrow (BM-MSC).Results: ScS-HOCl is close to d-ScS phenotype, leading to skin and lung fibrosis, together with anti-topoisomerase 1 antibody production. We show beneficial effects of a preventive or curative injection of syngeneic MSCs, reducing tissue fibrosis. Fibrosis reduction following MSC treatment involves immunosuppressive effects, tissue remodeling via metalloprotease activation, and anti-oxydant activity. Similar benefits are observed in allogeneic and xenogeneic settings. ASC display greater immunosuppressive and remodeling capacities than BM-MSCs.Discussion and conclusion: Our study demonstrates the anti-fibrotic effects of MSCs in a relevant preclinical model of ScS, mimicking diffuse and rapidly progressive ScS. Pleiotropic capabilities of MSCs, combining anti-inflammatory, remodeling and antioxidant properties, are promising for future clinical applications in this disease.Keywords: systemic sclerosis, mesenchymal stem cells, HOCl

Le cancer du sein est une pathologie hétérogène au plan clinique et au moins 5 sous-types moléculaires ont pu être définis sur la base de différences d’expression ARNm. Ces sous-types présentent des différences de profils d’anomalies génomiques et de méthylation des cytosines. Ces différences génétiques et épigénétiques s’expliqueraient par des types cellulaires d’origines distincts au sein de l’épithélium mammaire, toutefois, ceci n’a pas été confirmé clairement à ce jour. Alternativement, il a été proposé que l’activation de voies oncogéniques différentes pouvait avoir un impact significatif sur les modifications génétiques ou épigénétiques. Dans ce travail nous avons voulu vérifier cette hypothèse en l’appliquant à un modèle de cellules épithéliales mammaires normales primaires humaines, que nous avons isolé des à partir de glandes mammaires. Ces cellules ont été transformées en deux étapes par transduction avec (i) un shARN ciblant TP53, (ii) un oncogène. Nous avons sélectionné 3 oncogènes qui activent des voies de signalisations distinctes CCNE1, HRAS-v12 et WNT1. Nous avons établi un modèle de transformation tumorale en trois étapes, cellules normales, immortalisées et transformées, permettant de suivre les modifications moléculaires associées à chaque étape et de vérifier si l’activation de voies oncogéniques distinctes produisait des profils d’anomalies différents. Les différents modèles ont été analysés par CGH-array, RRBS, transcriptome et miRNA à des temps de culture définis.Nos résultats montrent que l’activation de la voie RAS aboutit à des profils d’anomalies génétiques et de méthylation des CpG radicalement différents de ceux obtenus après surexpression des gènes CCNE1 et WNT1. Ces différences apparaissent très rapidement après transduction des oncogènes alors que les profils des cellules CCNE1 et WNT1 divergent plus tardivement. Enfin, l’inactivation de p53 n’induit pas par elle-même une instabilité élevée, mais produit un contexte de plasticité favorable aux modifications génétiques et épigénétique.Par ailleurs, nous avons noté des différences phénotypiques entre les HMEC RAS (mésenchymateuses) et les HMEC CCNE1 et les HMEC WNT1 (épithéliales). Dans ce travail, je montre que les HMEC shp53 immortalisées présentent une plasticité phénotypique, une partie des cellules entrant en EMT spontanément, l’autre restant épithéliales. J’ai montré que la transduction RAS sélectionnait les cellules ayant effectué une EMT, alors que la transduction de CCNE1 ou WNT1 sélectionnait les cellules épithéliales. J’ai cherché à identifier les déterminants de ces changements phénotypiques et mes résultats suggèrent qu’ils résultent d’une balance entre une signalisation TGFB1/BMP1, qui favorise l’EMT, et BMP4/WNT7 qui favorise la MET. / Breast cancer is a heterogeneous pathology. Based on the differences of mRNA expression, at least five molecular subtypes have been defined. These subtypes show differences in profiles of genomic abnormalities and CpG methylation. These molecular subtypes are thought to originate from different cell lineages in the mammary gland. However, this has not yet been clearly demonstrated. Alternatively, it has been proposed that the activation of different oncogenic pathways could have a significant impact on genetic or epigenetic changes.We wanted to verify this hypothesis by applying it to a normal primary human mammary epithelial cells (HMEC) model, which we isolated from normal mammary explants. These cells were transformed in two step process by sequential transduction of (i) a shRNA targeting TP53, (ii) an oncogene. We selected 3 oncogenes that activate distinct signaling pathways CCNE1, HRAS-v12 and WNT1. Our tumor transformation model was established in three-step, normal, immortalized and transformed cells, allowing us to monitor the molecular changes associated with each step and to verify whether the activation of distinct oncogenic pathways produced different profiles of genetic and epigenetic modifications. The different models were analyzed at defined culture times by CGH-array, RRBS, transcriptome and miRNA. Our results show that genetic abnormalities and CpG methylation profiles are different between cells where the RAS pathway was activated and cells overexpressing WNT1 or CCNE1. These differences appear rapidly after oncogene transduction, whereas the profiles of the CCNE1 and WNT1 cells diverged later. Finally, inactivation of p53 by itself does not induce high instability, but produces a context of plasticity favorable to genetic and epigenetic changes.In addition, we noted phenotypic differences between HMEC RAS (mesenchymal) and HMEC CCNE1 and HMEC WNT1 (epithelial). In this work, I show that the immortalized HMEC shp53 exhibit a phenotypic plasticity, where some cells enter a spontaneous EMT and the others remain epithelial. I showed that RAS transduction selected cells that are undergoing an EMT, whereas transduction with CCNE1 or WNT1 selected the epithelial cells. I have sought to identify the determinants of these phenotypic changes and my results suggest that a balance exists between TGFβ1 / BMP1 signaling, which promotes EMT, and BMP4 / WNT7a which promotes TEM.

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Mark L. Weiss / Mesenchymal stromal cells (MSCs) derived from the umbilical cord (UC-MSCs) have therapeutic applications and are studied to understand their potential uses and immunomodulatory properties. Research must identify good manufacturing process (GMP) compliant methods to isolate and expand UC-MSCs. In addition, MSCs metabolism characteristics in culture are unknown, warranting further investigation. Viability of MSCs decreases after cryopreservation, which is detrimental to clinical translation. Previously published methods used to isolate MSCs from the umbilical cord included open dissection steps and xenogeneic components. Here, I developed improved methods by eliminating dissection which reduces contamination risks. Instead, I used the whole umbilical cord and Miltenyi dissociator tubes to mechanically and enzymatically dissociate cells in a closed system. Xenogeneic components were decreased by using medium containing pooled human platelet lysate instead of fetal bovine serum. The cell numbers isolated from umbilical cord averaged 2.68 x 10⁵ per cm, which represents greater than 20 fold improvement over the previous method. Moreover, expansion cell numbers were increased using 10% pooled human platelet lysate supplemented media. The UC-MSCs generated here met the International Society of Cell Therapy (ISCT) definition of MSCs. Metabolism characteristics of MSCs indicated that glucose was the critical metabolite, maintaining cells longer in culture than glutamine. Cell death followed depletion of glucose, too. Finally, the average viability after thawing cryopreserved MSCs was more than 95%, higher than previous methods. The improvements I introduced to our methodology could speed clinical translation of MSCs as an allogeneic cellular therapy

Increasing demands for organ transplants and the depleting supply of available organs has heightened the need for alternatives to this growing problem. Tissue engineers strive to regenerate organs in the future; however doing so requires a fundamental understanding of organ development and its key processes. The first chapter of this thesis provides a brief overview of developmentally inspired engineering, specifically in the context of how I approach this challenge in this thesis. The second chapter provides an in depth review of current and past work focused on organ regeneration from a developmentally-inspired perspective, and using tooth formation as a model system. The third chapter describes the design and fabrication of a thermoresponsive polymer inspired by an embryonic induction mechanism, and demonstrates its ability to induce tooth differentiation in vitro and in vivo. This is effectively a 3D `shrink wrap'-like polymer sponge that constricts when it is warmed to body temperature and induces compaction of cells contained within it, hence recapitulating the mesenchymal condensation process that has been shown to be a key induction mechanism that triggers formation of various epithelial organs, including tooth in the embryo. The fourth chapter describes the fabrication of a novel microarray screening platform consisting of a unique set of ECM proteins (collagen VI, tenascin, and combination of the two at different coating densities) on an array of soft substrates (~130-1500 Pa) that are physiologically relevant to the embryonic microenvironment. This technology demonstrated the capacity to analyze combinatorial effects of these ECM proteins and soft substrates on cell density, cell area and odontogenic differentiation in murine mandible embryonic mesenchymal cells. The fifth chapter of this thesis summarizes and discusses the advantages, limitations and future potential of the findings described in the previous two chapters in the context of organ engineering and regeneration. Taken together, the work and results presented in this thesis have led to the development of new insights, approaches and tools for studying organ formation and potentially inducing organ regeneration, which are inspired by key developmental mechanisms used during embryonic organ formation. / Engineering and Applied Sciences

Periodontal disease affects 47% of Americans over 30. Characterized by microbial dysbiosis and unregulated inflammation, severe periodontitis causes degradation of bone and soft tissue around teeth. Current treatments have limited regenerative outcomes and frequent reinfection by harmful bacteria. Human mesenchymal stem cells (hMSCs) have been shown to promote wound healing and tissue regeneration. Many therapeutic benefits of hMSCs are due to their secretome products, like exosomes. Our long-term goal is to develop periodontal therapies with hMSC exosomes. The objectives of this study were to determine the effect of hMSC-derived exosomes on cellular activity of hMSCs and investigate whether hMSC exosome treatment reduces pro-inflammatory cytokine production in LPS-activated RAW264.7 cells. The specific aims of this study were: 1) Determine the characteristics of hMSC-derived exosomes, 2) Determine the biological effect of exosomes on cellular activity of hMSCs, 3) Determine whether exosomes treatment can inhibit cytokine production in activated RAW264.7 cells, and 4) Determine the role of exosomal miRNA in pro-inflammatory cytokine production of RAW264.7 cells. To investigate, exosomes were first harvested from hMSCs culture media through ultracentrifugation. Exosomes were then observed under a transmission electron microscope (TEM) and assessed for surface markers using Western Blot. A transwell migration assay was used to evaluate the chemotactic effect of exosomes. To study the effect of exosomes on stem cell proliferation, exosomes were administered to hMSCs. The immunogenicity of MSC exosome was also evaluated. After 72 hours, cells were lysed and DNA was measured. To study anti-inflammatory effects of exosomes, LPS stimulated RAW264.7 cells were treated with exosomes. Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) levels of supernatant were measured by ELISA. To study exosomal miRNA, exosomal miRNAs were overexpressed in RAW264.7 cells and these cells were stimulated with LPS. IL-6 and TNFα were measured by ELISA. TEM images showed that exosomes are nano-sized vesicles (~100 nm). Western blot images showed that CD63 and CD81 are enriched in exosomes compared to total cell lysates. Exosome treatment increased cell proliferation and migration in hMSCs. At the doses that are chemotactic and mitogenic, MSC exosomes had minimal effect on the inflammatory cytokine IL-6 production. Treatment with exosomes significantly decreased IL-6 and TNFα production in RAW264.7 cells activated by LPS. Transfecting RAW264.7 cells with exosomal miR-760 significantly decreased IL-6 production, but had minimal effect on TNFα. Our results indicate that exosomes have a pleiotropic activity, which includes stimulating stem cell migration and proliferation, and mitigating the inflammatory response. Therefore, hMSC exosome delivery is promising for the treatment of periodontal diseases.

Cellulose nanowhiskers (CNWs) are high aspect ratio rod-like nanoparticles with diameters on the order of a few nanometers. For the very first time CNWs are demonstrated as a useful material for guided tissue engineering. Due to their nanoscale dimensions and high aspect ratio, highly oriented spin coated surfaces of CNWs are shown to direct the morphology and terminal differentiation of myoblasts, allowing the culture of skeletal muscle-like tissue with a more physiologically relevant structure.CNWs are prepared from cellulose extracted from the tunicate Ascidiella sp. using acid hydrolysis to prepare high aspect ratio particles with diameters of approximately 5 to 6 nm. A spin coating method is used to prepare sparsely adsorbed sub-monolayers of CNWs with a high degree of relative orientation. The surfaces have a mean feature height of only 5.5 nm and the degree of CNW adsorption and orientation is modulated by altering the preparation procedure. When C2C12 myoblasts are seeded to the surfaces, the cells adopt highly oriented morphologies induced by the CNWs via contact guidance. This is a demonstration of contact guidance on some of the smallest topographical features ever reported. Furthermore, the highly oriented CNWs promote fusion and terminal differentiation of the myoblasts to form multinucleated myotubes with a striking degree of parallel orientation.CNW surfaces are also shown to support the adhesion and spreading of human mesenchymal stem cells, inducing the adoption of highly oriented cell morphologies. The ability of hMSCs to undergo cell fusion with C2C12 myotubes highlights the great potential for tissue engineering human skeletal muscle, using CNWs to direct the structure of the tissue. The bioactivity and low cytotoxicity of CNWs, coupled with their low cost and simple production procedure, indicates that CNWs will be a useful material for tissue engineering and regenerative medicine.

In the late stages of cancer, tumors acquire the ability to spread throughout the body and invade distant tissues in a process called metastasis. Studies have shown that metastasis is responsible for 90% of all cancer-related deaths, making this an important field of study. In breast cancers, 30% of patients overexpress the HER2 oncoprotein, causing a more invasive and metastatic disease. Invasion can be stimulated in vitro using the soluble ligand transforming growth factor-β (TGFβ) to induce a process called EMT (epithelial-to-mesenchymal transition), where epithelial cells transition into a migratory phenotype through cell-cell junction breakdown. SLK is a Ste20-like kinase that has been linked to many processes, including cell migration and signaling downstream of the HER2 receptor complex. Here we show that the cellular migration and invasion of TGFβ-treated normal mammary epithelial cells is significantly impaired in the absence of SLK. Additionally, immunofluorescence analyses demonstrate that SLK knockdown conditions decrease a cell’s ability to progress through EMT due to the visible staining of epithelial markers. We find that SLK-depleted cultures express significantly lower levels of Snail1,and fibronectin mRNA levels following TGF-β treatment. Surprisingly, our data demonstrates that SLK kinase activity is not activated downstream of TGF-β stimulation, and that a kinase-dead SLK rescues Snail1 mRNA expression levels. Together these data suggest that SLK plays a novel role in TGFβ-induced epithelial-to-mesenchymal transition in a kinase activity-independent manner.