Les cancers, qu’il s’agisse de leucémies ou de tumeurs solides, sont le résultat de proliférations cellulaires anormales et non contrôlées au sein des tissus. Ces proliférations anarchiques sont le reflet d’une surexpression et/ou sur-activation de protéines intracellulaires engendrées par un événement oncogénique. Aujourd’hui encore il est donc nécessaire de trouver de nouvelles molécules à usage thérapeutique ciblant spécifiquement ces protéines. C’est dans ce contexte que les facteurs de transcription STAT5 constituent de véritables cibles de choix puisque ces protéines participent activement à la leucogénèse. L’implication directe des protéines STAT5 dans la génèse des leucémies a été démontrée par l’utilisation de formes mutées constitutivement active de STAT5. Les facteurs de transcription STAT5 jouent un rôle essentiel dans la voie de signalisation JAK/STAT. Cette voie aboutit à la régulation de grandes fonctions biologiques telles que la prolifération cellulaire, la différenciation cellulaire ou encore l’apoptose. L’objectif de ce projet consiste donc à cibler spécifiquement les protéines STAT5 dans le but de rétablir le processus de mort cellulaire et empêcher la prolifération des cellules cancéreuses. Les inhibiteurs spécifiques des protéines STAT5 sont sélectionnés selon la méthode SELEX qui permet d’isoler des ligands structurés de forte affinité pour la protéine. L’affinité et la spécificité de ces inhibiteurs, appelés aptamères, sont caractérisées à partir de modèles cellulaires de leucémies dépendant de l’activité des facteurs de transcription STAT5. Les aptamères sont aujourd’hui de véritables outils thérapeutiques en pleine évolution. / Leukemias are due to abnormal cell proliferation, which is the result of intracellular over-expression or excessive activation of protein due to oncogenic event. Still today, it is necessary to find new therapeutic molecules, which specifically target these proteins. STAT5, via the JAK/STAT signaling pathway, controls fundamental cellular processes, including .cell survival, proliferation and differentiation. To struggle against tumorigenesis, JAK/STAT signaling pathway has to be inhibited. The aim of this project is to target specifically STAT5 factors to restore healthy signal transduction. We generated aptamers by an iterative in vitro selection. Aptamers are short-structured single strand DNAs or RNAs that bind with high affinity and specificity to their target. Once STAT5B recombinant proteins are produced, they are subjected to SELEX process. The number of rounds depends on various parameters. After seven rounds, two sequences are retrieved. The specificity and affinity of these aptamers are assessed by fluorescent immunoassays. Binding affinity and kinetics of interaction are characterized by SPR. Aptamer anti proliferative effects are determined by evaluation of the growth of cells depending on STAT5. Finally, we developed several .assays aiming at understanding the mechanism of an aptamer action on STAT5B such as phosphorylation measurement and EMSA. Aptamers are now emerging therapeutic tools; they exhibit significant advantages relative to protein therapeutics.

Differenzierungsentscheidungen von Zellen werden durch die Integration mehrerer Stimuli bestimmt. Die Differenzierung von Helfer-T-Zellen (Th-Zellen) ist hierfür ein gut untersuchtes Beispiel: reife Th-Zellen entwickeln sich beim Kontakt mit einem für sie spezifischen Antigen zu einem spezialisierten Subtyp, der von den in ihrer Umgebung vorhandenen Zytokinen abhängt und exprimieren dann einen spezifischen Mastertranskriptionsfaktor. Die häufigsten Th-Zell-Subtypen sind T-bet-exprimierende Th1-Zellen und GATA-3-exprimierende Th2-Zellen. Neuere Entdeckungen bezüglich der Plastizität von Th-Zell-Subtypen sowie die Existenz von T-bet+GATA-3+ Hybrid-Phänotypen haben die detaillierte Untersuchung vom Differenzierungsprozessen von Th-Zellen mit komplexer Zytokinsignale motiviert. Dazu haben wir systematisch die Zytokine IFN-g, IL-12 und IL-4 während der primären Differenzierung Th-Zellen titriert und Signaltransduktion und Zielgenexpression quantifiziert. Der Umfang und die Komplexität der Daten machten eine systematische Analyse notwendig, um involvierte Mechanismen genau zu identifizieren. Lineare Regressionsanalyse wurde verwendet, um die Netzwerktopologie zu extrahieren, wobei schon bekannte und zahlreiche neue Interaktionen vorausgesagt wurden. Die prognostizierte Netzwerktopologie wurde dann verwendet, um ein mechanistisches, mathematisches Modell der Zytokinsignalintegration zu entwickeln. Diese Methode hat ein hochgradig vernetztes regulatorisches Netzwerk inferiert. Bisher nicht beschriebene Funktionen von STAT-Proteine, die die Neuverkabelung des Netzwerkes während der Differenzierung vermitteln, wurden vorhergesagt. Ausgewählte neue Interaktionen wurden in gezielten genetischen Experimenten bestätigt. Während gegenseitige Inhibitionsmotive oft als kanonische digitale Schalter interpretiert werden, funktioniert das Th-Zell-Netwerk als ein Rheostat, der Variationen der Zytokinsignale in graduelle Expressionsänderungen der Mastertranskriptionsfaktoren übersetzt. Unsere Arbeit erklärt mechanistisch das beobachtete Kontinuum von Th-Zelldifferenzierungszuständen entlang der Th1-Th2-Achse und beschreibt eine quantitative Methode für die datenbasierte Inferenz zellulärer Netzwerke der Signalintegration. / Cell-fate decisions are governed by the integration of multiple stimuli. Th cell differentiation is a well-studied example thereof: mature Th cells differentiate into a specialised subtype upon encounter with their cognate antigen depending on the polarising cytokines present in their environment and start expressing specific master transcription factors. The most common Th cell subtypes are T-bet-expressing Th1 cells and GATA-3-expressing Th2 cells. Recent discoveries concerning the plasticity of Th cell subtypes as well as the existence of stable T-bet+GATA-3+ hybrid Th1/2 phenotypes have stimulated the detailed study of the differentiation process under different assumptions than the hitherto valid paradigm of single master transcription factor expression by using complex cytokine signals as inputs. Here, we developed a data-based approach for inferring the molecular network underlying the differentiation of T-bet- and/or GATA-3 expressing lymphocytes. We performed systematic titrations of the polarising cytokines IFN-g, IL-12 and IL-4 during primary differentiation of Th cells and quantified signal transduction as well as target-gene expression. The size and complexity of the dataset made a systematic analysis necessary to identify the mechanisms involved. To extract the network topology, we used linear regression analysis, retrieving known regulatory mechanisms and predicting numerous novel ones. This network topology was used to develop a mechanistic mathematical model of cytokine signal integration. This approach inferred a highly connected regulatory network. Previously undescribed functions of STAT proteins mediating network rewiring during differentiation were predicted. Selected new interactions were confirmed by experiments using gene-deficient cells. Importantly, while mutual-inhibition motifs are often considered canonical digital switches, the inferred Th-cell network acts as a rheostat, generating a continuum of differentiated states along the Th1-Th2 axis. This work explains the observed Th1-Th2 cell fate continuum mechanistically and provides a quantitative framework for the data-based inference of cellular signal integration networks.

Mouse embryonic stem cells (mESCs) can be maintained in a proliferative and undifferentiated state over many passages (self-renewal) while retaining the potential to give rise to every cell type of the organism (pluripotency). Autocrine FGF4/Erk signalling has been identified as a major stimulus for fate decisions and lineage commitment in these cells. Recent findings on serum-free culture conditions with specific inhibitors (known as 2i) demonstrate that the inhibition of this pathway reduces transcription factor heterogeneity and is vital to maintain ground state pluripotency of mESCs. We suggest a novel mathematical model to explicitly integrate FGF4/Erk signalling into an interaction network of key pluripotency factors (namely Oct4, Sox2, Nanog and Rex1). The envisaged model allows to explore whether and how proposed mechanisms and feedback regulations can account for different expression patterns in mESC cultures. We demonstrate that an FGF4/Erk-mediated negative feedback is sufficient to induce molecular heterogeneity with respect to Nanog and Rex1 expression and thus critically regulates the propensity for differentiation and the loss of pluripotency. Furthermore, we compare simulation results on the transcription factor dynamics in different self-renewing states and during differentiation with experimental data on a Rex1GFPd2 reporter cell line using flow cytometry and qRT-PCR measurements. Concluding from our results we argue that interaction between FGF4/Erk signalling and Nanog expression qualifies as a key mechanism to manipulate mESC pluripotency. In particular, we infer that ground state pluripotency under 2i is achieved by shifting stable expression pattern of Nanog from a bistable into a monostable regulation impeding stochastic state transitions. Furthermore, we derive testable predictions on altering the degree of Nanog heterogeneity and on the frequency of state transitions in LIF/serum conditions to challenge our model assumptions.

The availability of several methods to unambiguously mark individual cells has strongly fostered the understanding of clonal developments in hematopoiesis and other stem cell driven regenerative tissues. While cellular barcoding is the method of choice for experimental studies, patients that underwent gene therapy carry a unique insertional mark within the transplanted cells originating from the integration of the retroviral vector. Close monitoring of such patients allows accessing their clonal dynamics, however, the early detection of events that predict monoclonal conversion and potentially the onset of leukemia are beneficial for treatment. We developed a simple mathematical model of a self-stabilizing hematopoietic stem cell population to generate a wide range of possible clonal developments, reproducing typical, experimentally and clinically observed scenarios. We use the resulting model scenarios to suggest and test a set of statistical measures that should allow for an interpretation and classification of relevant clonal dynamics. Apart from the assessment of several established diversity indices we suggest a measure that quantifies the extension to which the increase in the size of one clone is attributed to the total loss in the size of all other clones. By evaluating the change in relative clone sizes between consecutive measurements, the suggested measure, referred to as maximum relative clonal expansion (mRCE), proves to be highly sensitive in the detection of rapidly expanding cell clones prior to their dominant manifestation. This predictive potential places the mRCE as a suitable means for the early recognition of leukemogenesis especially in gene therapy patients that are closely monitored. Our model based approach illustrates how simulation studies can actively support the design and evaluation of preclinical strategies for the analysis and risk evaluation of clonal developments.

Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone growth can be impaired due to disease, such as osteoporosis. Currently, intermittent parathyroid hormone (PTH) treatment is the only approved therapy in the United States for anabolic bone growth in osteoporosis patients. The anabolic effects of PTH treatment are due, at least in part, to modulation of the Wnt/β-catenin pathway. Activation of the Wnt/ β-catenin pathway using a small molecule inhibitor of GSK3β was previously shown to increase markers of bone formation in vitro. Our study utilized a zebrafish model system to study Wnt activated fin regeneration and bone growth. Wnt signaling is the first genetically identified step in fin regeneration, and bony rays are the main structure in zebrafish fins. Thus, zebrafish fin regeneration may be a useful model to study Wnt signaling mediated bone growth. Fin regeneration experiments were conducted using various concentrations of a GSK3β inhibitor compound, LSN 2105786, for different treatment periods and regenerative outgrowth was measured at 4 and 7 days post amputation. Experiments revealed continuous low concentration (4-5 nM) treatment to be most effective at increasing regeneration. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. In situ hybridization experiments were performed to examine effects of GSK3β inhibitor on Wnt responsive gene expression. Experiments showed temporal and spatial changes on individual gene markers following GSK3β inhibitor treatment. Additionally, confocal microscopy and immunofluorescence labeling data indicated that the Wnt signaling intracellular signal transducer, β-catenin, accumulates throughout GSK3β inhibitor treated tissues. Finally, experiments revealed increased cell proliferation in fin regenerates following LSN 2105786 treatment. Together, these data indicate that bone growth in zebrafish fin regeneration is improved by activating Wnt signaling. Zebrafish Wnt signaling experiments provide a good model to study bone growth and bone repair mechanisms, and may provide an efficient drug discovery platform.

Indiana University-Purdue University Indianapolis (IUPUI) / The epigenetic state of any cell is, in part, regulated by the interaction of DNA with nuclear histones. Histone tails can be modified in a number of ways that impact on the availability of DNA to interact with transcriptional complexes, including methylation, acetylation, phosphorylation, ubiquituination, and sumoylation. Histones are acetylated by a large family of enzymes, histone acetyl transferases (HATs), and deacetylated by the histone deacetylases (HDACs). Acetylated histones are generally considered markers of genomic regions that are actively being transcribed, whereas deacetylated and methylated histones are generally markers of regions that are inactive. The goal of the present study was to 1) study the epigenetic state with regard to the presence of euchromatin and heterochromatin in the developing chick and murine retina, 2) study and compare the localization patterns of the classical HDACs in the developing chick and murine retina with respect retinal progenitors and early differentiated cell types 3) to test the hypothesis that overall HDAC activity is required for dividing retinal progenitors to leave the cell cycle and differentiate. Our results showed that the classical HDACs were ubiquitously expressed in the developing chick and murine retinas. Species specific differences as well as stage dependent variations were observed in the localization of the HDACs in the cell types that were studied in the chick and murine retina. Our preliminary results also showed that HDAC inhibition may lead to the inability of the cell types to leave the cell cycle and a subsequent increase in the number of progenitor cells present in the developing chick retina.

Indiana University-Purdue University Indianapolis (IUPUI) / Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial Colony Forming Cells (ECFCs) are highly proliferative endothelial progenitor cells with clonal proliferative potential and in vivo vessel forming ability. While endothelial cells have been derived from human induced pluripotent stem cells (hiPS) or human embryonic stem cells (hES), they are not highly proliferative and require ectopic expression of a TGFβ inhibitor to restrict plasticity. Neuropilin-1 (NRP-1) has been reported to identify the emergence of endothelial precursor cells from human and mouse ES cells undergoing endothelial differentiation. However, the protocol used in that study was not well defined, used uncharacterized neuronal induction reagents in the culture medium, and failed to fully characterize the endothelial cells derived. We hypothesize that NRP-1 expression is critical for the emergence of stable endothelial cells with ECFC properties from hES cells. We developed a novel serum and feeder free defined endothelial differentiation protocol to induce stable endothelial cells possessing cells with cord blood ECFC-like properties from hES cells. We have shown that Day 12 hES cell-derived endothelial cells express the endothelial markers CD31+ NRP-1+, exhibit high proliferative potential at a single cell level, and display robust in vivo vessel forming ability similar to that of cord blood-derived ECFCs. The efficient production of the ECFCs from hES cells is 6 logs higher with this protocol than any previously published method. These results demonstrate progress towards differentiating ECFC from hES and may provide patients with stable autologous cells capable of repairing injured, dysfunctional, or senescent vasculature if these findings can be repeated with hiPS.

Indiana University-Purdue University Indianapolis (IUPUI) / The inner ear contains specialized sensory epithelia that detect head movements, gravity and sound. Hearing loss and imbalance are primarily caused by degeneration of the mechanosensitive hair cells in sensory epithelia or the sensory neurons that connect the inner ear to the brain. The controlled derivation of inner ear sensory epithelia and neurons from pluripotent stem cells will be essential for generating in vitro models of inner ear disorders or developing cell-based therapies. Despite some recent success in deriving hair cells from mouse embryonic stem (ES) cells, it is currently unclear how to derive inner ear sensory cells in a fully defined and reproducible manner. Progress has likely been hindered by what is known about induction of the nonneural and preplacodal ectoderm, two critical precursors during inner ear development. The studies presented here report the step-wise differentiation of inner ear sensory epithelia from mouse ES cells in three-dimensional culture. We show that nonneural, preplacodal and pre-otic epithelia can be generated from ES cell aggregates by precise temporal control of BMP, TGFβ and FGF signaling, mimicking in vivo development. Later, in a self-guided process, vesicles containing supporting cells emerge from the presumptive otic epithelium and give rise to hair cells with stereocilia bundles and kinocilium. Remarkably, the vesicles developed into large cysts with sensory epithelia reminiscent of vestibular sense organs (i.e. the utricle, saccule and crista), which sense head movements and gravity in the animal. We have designated these stem cell-derived structures inner ear organoids. In addition, we discovered that sensory-like neurons develop alongside the organoids and form putative synapses with hair cells in a similar fashion to the hair cell-to-neuron circuit that forms in the developing embryo. Our data thus establish a novel in vitro model of inner ear organogenesis that can be used to gain deeper insight into inner ear development and disorder.