No description available.

Articular cartilage lines the surfaces of load bearing joints and has limited capabilities for self-repair due to its alymphatic and avascular structure. Attempts at making repairs to this tissue has resulted in substandard materials and/or causing further injury to the patient making this tissue a prime candidate for tissue engineering studies incorporating adult stem cells. These studies have given rise to some answers and many more questions including a search for alternative stem cell sources and what biochemical changes the cells undergo during the differentiation of these stem cells into chondrocytes, the cells which make up articular cartilage. Recently, periodontal dental ligament stem cells (PDLs) have come to the forefront as a practical alternative to other adult stem cells as well as the involvement of the mitogen-activated protein kinases (MAPKs) in stem cell differentiation via mechanical stimulation. During dynamic unconfined compression, levels of p42/44 MAPK increased by 50% (p<0.05). Additionally, the expression of the chondrogenic differentiation factor SRY (sex determining region Y)-box 9 (SOX-9) increased by 3-fold (p<0.05) as well as the chondrocyte marker aggrecan by over 2-fold after 4h of dynamic unconfined compression. Addition of the p42/44 phosphorylation inhibitor PD98059, along with compression, yielded no change in SOX-9 or aggrecan expression levels from basal levels in uncompressed controls. Inhibition of p38 MAPK or JNK phosphorylation during unconfined compression had no effect on the elevated expression of SOX-9 and aggrecan as compared to compressed cells without the addition of an inhibitor. It is therefore the overall findings of this study that PDLs possess the ability to differentiate into chondrocytes by mechanical compression and this differentiation is mediated by the p42/44 MAPK cascade.

Transforming growth factor (TGF) β is a tumor suppressor during early tumor development, by inhibiting proliferation and inducing apoptosis. At later stages of cancer, it becomes a tumor promoter, and promotes tumor cell migration and invasion. TGFβ signals via its type II and type I receptors to several downstream signaling pathways. In the present work we have focused on the TRAF6 (tumor necrosis factor receptor-associated factor 6)/ TAK1 (TGFβ activated kinase 1) signaling pathway and the Smad7-dependent activation of p38 in prostate carcinoma cells (PC3U). We found that TGFβ-induced activation of the ubiquitin ligase TRAF6 was needed for cell invasion, by a mechanism that involves activation of the metalloproteinase TNFα converting enzyme (TACE), via protein kinase Cζ (PKCζ). TACE cleaves the TβRI, whereafter the intracellular domain (ICD) translocates to the nucleus, where it binds to the transcriptional co-activator p300 and regulates gene expression, promoting invasion. Interestingly, the translocation of the TβRI ICD was observed in several cancer cell lines and in sections of primary tumors, but not in primary prostate epithelial cells. We also found that Smad7 and adenomatous polyposis coli (APC) are important for TGFβ- and epidermal growth factor (EGF)-induced cell migration in PC3U cells. TGFβ induces the formation of a complex consisting of Smad7, p38, glycogene synthase kinase 3β (GSK-3β), APC and β-catenin, which localizes to the membrane ruffles in the leading edge of migrating cells. The complex links the TβRI to the microtubule system and promotes membrane ruffling and microtubule polarization, which are known to be important for cell migration. In the EGF signaling pathway, Smad7 was found to be important for phosphorylation of the EGF receptor at Tyr1068, for the activation of p38 and JNK, and for induction of membrane ruffles. Smad7 is required for TGFβ-induced activation of p38 and apoptosis. We found that Smad7 forms a complex with p38 and ataxia telangiectasia mutated (ATM), which is important for activation of p53 mediated apoptosis. Many tumor cells including the PC3U cells lack a functional p53, which is one of the reasons to why cancer cells can avoid the tumor suppressor effects of TGFβ.

Articular cartilage functions to maintain joint mobility. The loss of healthy, functional articular cartilage due to osteoarthritis or injury can severely compromise quality of life. To address this issue, cartilage tissue engineering approaches are currently in development. Bone marrow-derived mesenchymal progenitor cells (MPCs) hold much promise as an alternative cell source for cartilage tissue engineering. While previous studies have established that MPCs from humans and multiple other species undergo in vitro chondrogenic differentiation, additional research is needed to define conditions that will enhance MPC differentiation, increase matrix production by differentiating cultures, and support development of functional tissue-engineered cartilage constructs. Mechanical loading may be an important factor regulating chondrogenic differentiation of MPCs and cartilage matrix formation by chondrogenic MPCs. This thesis work evaluated the influence of oscillatory unconfined compressive mechanical loading on in vitro MPC chondrogenic activity and biosynthesis within hydrogel suspension. Loading was conducted using MPCs cultured in media supplements supporting chondrogenic differentiation. Possible interactions between the number of days in chondrogenic media preceding loading initiation and the ability of the MPC culture to respond to mechanical stimulation were explored in two different loading studies. The first loading study investigated the effects of 3 hour periods of daily oscillatory mechanical stimulation on subsequent chondrogenic activity, where chondrogenic activity represented an assessment of cartilage matrix production by differentiating MPCs. This study found that oscillatory compression of MPCs initiated during the first seven days of culture did not enhance chondrogenic activity above the level supported by media supplements alone. The second loading study evaluated changes in biosynthesis during a single 20 hour period of oscillatory mechanical stimulation to assess mechanoresponsiveness of the MPC cultures. This study found that MPCs modulated proteoglycan and protein synthesis in a culture time-dependent and frequency-dependent manner upon application of oscillatory compression. Together the two loading studies provide an assessment of dynamic compressive mechanical loading influences on MPC cultures undergoing chondrogenic differentiation. The information gained through in vitro studies of differentiating MPC cultures will increase basic knowledge about progenitor cells and may also prove valuable in guiding the future development of cartilage tissue engineering approaches.

Cellular fate, once established, is usually stable for the lifetime of the cell. However, the mechanisms that restrict the developmental potential of differentiated cells are in principle reversible, as demonstrated by the success of animal cloning from a somatic genome through somatic cell nuclear transfer (SCNT). An increased understanding of the molecular determinants of cell fate has also enabled the reprogramming of cell fate using defined transcription factors; recently, these efforts have culminated in the discovery of four genes that convert somatic cells into induced pluripotent stem cells (iPSCs), which resemble embryonic stem cells (ESCs) and can give rise to all the cell types in the body. As a first step toward generating clinically useful iPSCs, we identified a small molecule, RepSox, that potently and simultaneously replaces two of the four exogenous reprogramming factors, Sox2 and cMyc. This activity was mediated by the inhibition of the Transforming Growth Factor-\(\beta\) \((Tgf-\beta)\) signaling pathway in incompletely reprogrammed intermediate cells. By isolating these stable intermediates, we showed that RepSox acts on them to rapidly upregulate the endogenous pluripotency factor, Nanog, allowing full reprogramming to pluripotency in the absence of Sox2. We also explored lineage conversion as an alternative approach for producing a target cell type in a patient-specific manner, without first generating iPSCs. A combination of pro-neural as well as motor neuron-selective factors could convert fibroblasts directly into spinal motor neurons, the cells that control all voluntary movement. The induced motor neurons (iMNs) displayed molecular and functional characteristics of bona fide motor neurons, actuating muscle contraction in vitro and even engrafting in the developing chick spinal cord when transplanted. Importantly, functional iMNs could be produced from fibroblasts of adult patients with the fatal motor neuron disease, amyotrophic lateral sclerosis (ALS). Given the therapeutic value of generating patient-specific cell types on demand, defined-factor reprogramming is likely to serve as an important tool in regenerative medicine. It is hoped that the different approaches presented here can complement existing technologies to facilitate the study and treatment of intractable human disorders.

Objective: Endothelial cell (EC)-induced mesenchymal cell (MC) differentiation toward a mural cell phenotype requires transforming growth factor beta (TGF-β), cell contact and connexin 43 (Cx43)- or Cx45- heterocellular gap junction intercellular communication (GJIC). However, the identity of the communicated signal, the features of Cx43 required, and the possible regulatory mechanisms have not been elucidated and were investigated herein. Methods & Results: To determine whether channel functionality and the major regulatory domain (the carboxyl terminus, CT) of connexin Cx43 are necessary to support EC-induced differentiation, Cx43 deficient MCs (incapable of undergoing EC-induced mural cell differentiation without re-expression of Cx43 or Cx45) were transduced with wild-type (Cx43wt), channel dead, or truncated (Cx43tr-residues 258-382 deleted) versions of Cx43 and their ability to support EC-induced differentiation was assessed. Our data indicate that both channel functionality and presence of the CT domain are both necessary for EC-induced mural cell differentiation. Moreover, expression of Cx40 did not restore ability of MCs to undergo EC-induced mural cell differentiation, despite supporting GJIC. To determine whether (and which) specific regulatory sites in the carboxyl terminus are necessary for EC-induced mural cell differentiation, constructs of Cx43 with serine to alanine substitutions at the mitogen activated protein kinase (MAPK) or protein kinase C (PKC) target sites were introduced into Cx43 deficient MCs and their ability to undergo EC-induced differentiation was tested. The data indicated that the MAPK targeted serines (S255,279,2982) are necessary, while the PKC targeted serine (S368) is dispensable, for this process. To determine whether calcium ions might be the messengers communicated between ECs and MCs, we investigated whether elevation in EC free intracellular calcium concentration (with ionomycin treatment) can replace Cx43-mediated GJIC, activate TGF-β and induce differentiation. Conclusions: Channel functionality, CT domain and the MAPK target sites in Cx43 are all necessary, and neither alone is sufficient, for Cx43-mediated, EC-induced mural cell differentiation. Unlike Cx43, Cx40 is not capable of supporting EC-induced differentiation, despite supporting GJIC. Calcium is unlikely to be the messenger critical to TGF-β activation during EC-induced differentiation, but similar signaling pathways can be initiated. Taken together, these data support a role for connexins in EC-induced differentiation that is complex and goes beyond that of a simple conduit.

The Snail1 transcriptional repressor is a key factor responsible in triggering epithelial to mesenchymal transition. Although Snail1 is widely expressed in early development, it is limited in adult animals to a subset of mesenchymal cells where it has a largely unknown function. In this project we have demonstrated that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to TGF-[beta]1 which showed a strong Snail1 dependence. Snail1-depletion in conditional knock-out adult animals caused a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiated to osteoblasts or adipocytes and, in contrast to controls, were resistant to the TGF-[beta]1-induced differentiation block. TGF-[beta]1 was unable to up-regulate most of its targets in Snail1 KO MSCs, an effect that was related, but not limited, to defective PTEN repression and Akt activation. Correspondingly, an analysis of human sarcomas also showed enhanced expression of Snail1 in undifferentiated tumors, which was strongly associated with high expression of TGF-[beta] and poor outcome. These results not only demonstrate a new role for Snail1 in TGF-[beta] response and MSC maintenance but also suggest the involvement of MSCs in sarcoma generation. / El repressor transcripcional Snail1 ha estat descrit principalment com el responsable de la inducció de la transició epiteli mesènquima. Encara que Snail1 s’expressa durant les etapes més primerenques del desenvolupament embrionari, la seva expressió en adults es veu limitada en un conjunt de cèl•lules mesenquimals sense saber-se la seva funció. En aquest projecte hem demostrat que Snail1 es requereix per mantenir el fenotip més indiferenciat de les cèl•lules mare del mesènquima. Aquesta funció la fa en part, per la capacitat de resposta de la citoquina TGF-[beta] la qual mostra una força dependència amb Snail1. Quan s’elimina Snail1 en ratolins adults provoca una clara disminució en el nombre de cèl•lules mare de la medul•la òssia. Aquestes cèl•lules en cultiu presenten una clara diferenciació prematura a osteoblasts i adipòcits. Pel contrari, tractaments amb TGF-[beta]1 aturen la diferenciació. El TGF-[beta]1 es incapaç de incrementar moltes dianes en cèl•lules mare del mesènquima aïllades del ratolí deficient per snail1, aquest efecte en part es degut a la repressió de PTEN i l’activació de AKT. L’anàlisi de sarcomes humans ens ha mostrat una alta expressió de Snail1, el qual també es troba associada amb una alta expressió de TGF-[beta] i baixa supervivència. Aquests resultats no només demostren una nova funció per Snail1 en resposta a TGF-[beta] i el manteniment de les MSC, sinó que també suggereix que Snail1 podria participar en la generació del sarcoma.

Chronic inflammatory conditions such as Crohn's disease (CD) and Ulcerative colitis (UC) are risk factors for colon cancer. TGFβ has been shown to be dysregulated in colon cancer. Bacteria-induced inflammation is necessary for the induction of colon cancer in TGFβ mouse models. However, the mechanism by which TGFβ regulates the inflammatory response in these models is not well elucidated. It was our thought that we needed to be able to distinguish what was TGFβ dependent and what was inflammation dependent. To do this we created 2 colonies of Smad3 mice. One colony was housed with normal colonic bacteria (Smad3-uninfected animals) and the other colony (Smad3-infected animals) had chronic H. hepaticus infection. As previously seen the Smad3⁻/⁻- infected animals developed colitis and carcinoma (~40%). In the absence of H. hepaticus infection SMAD3 was found to negatively regulate TLR4 expression. This was then exacerbated with the addition of H. hepaticus resulting extreme up-regulation of TLR4 and the downstream effectors IRAK4 and NF-κB in Smad3⁻/⁻-infected colonic tissues. Examination of adaptive immune regulation in this model demonstrated that SMAD3 was necessary for FOXP3 expression in H. hepaticus-infected splenocytes. Loss of SMAD3 resulted in up-regulation of IL17 and reduced iTreg populations. These data demonstrate the important role SMAD3 has in maintaining tolerance to microbial populations through both the innate and adaptive immune systems.

Breast cancer is comprised of several subtypes that are different from one another and thedivergence leads to different outcomes of the disease. There are known prognostic factors andphenotypic distinction in different biological factors and expression patterns, such as theestrogen receptor (ER), progesterone receptor (PR), Ki67, HER2/neu expression (HER2). Ingeneral, there are three breast cancer subtypes with the most recurring subtype being luminalA, and the other two being luminal B and triple negative breast cancer. Triple negative breastcancer is a heterogeneous subtype which is defined by the lack of expression of ERα, PR andHER2. Triple negative breast cancers are also very aggressive and have the worst prognosiscompared to the other two ERα positive tumors. The luminal A subtype can develop into ametastatic cancer thanks to the so-called epithelial-mesenchymal transition (EMT), whichaffects a subpopulation of epithelial cancer cells. EMT is the name of a process that takesplace during the embryonic development, the wound healing and cancer metastasis, where theepithelial cells will transform into mesenchymal cells which have higher invasive andmigratory properties. EMT occurs when epithelial cells lose their apical-basal polarity andthen the adherens- and tight junctions are dissolved. The adherens junction dissolution can beobserved as a downregulation of CDH1 (E-cadherin), which is regularly measured in EMTstudies. Many signaling pathways are associated with the promotion and establishment ofEMT e.g. transforming growth factor β (TGFβ), Notch and Wnt signaling. Bioinformaticscreening was performed to look for mRNA expression levels of ZEB1 and Snail1 indifferent breast cancer cell lines. By using chromatin immunoprecipitation-sequencing (ChIPSeq)in the triple negative (ER-, PR- HER2-) Hs578T breast cancer cell line, a genome-widescreen for ZEB1 and Snail1 binding sites had been performed before the start of the project.The Hs578T cell line expresses many of the EMT transcription factors that are relevant forthe project. Since the signaling of TGFβ is crucial for these genes, manipulation of thissignaling pathway is needed to be able to analyse its importance for the function of thesegenes. To inhibit the activity of TGFβ, the small molecule GW6604 was used to inhibit theTGFβ type I receptor kinase (TβRI) and in that way inhibiting the signaling from thisreceptor. In addition, ZEB1 and Snail1 were knocked out by the use of the transfection andCRISPR/Cas9 knockout technique. By investigating mRNA and protein levels of chosengenes in both control Hs578T cells and ZEB1 and Snail1 knockout Hs578T cells, up or downregulation of some of these genes can be seen with stimulation with TGFβ. The knockout ofSnail1 but not of ZEB1 indicated that the loss of Snail1 generated breast cancer cells thatcould try to revert to epithelial at the phenotypic level.

Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.