Yes / Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2-glia. NG2-glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2-glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2-glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2-glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti-NG2 antibody in combination with secondary saporin-conjugated antibody selectively kills NG2-expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2-glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2-glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.

Yes / The maintenance of a proper nuclear architecture and 3D organization of the genes, enhancer elements and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by marked decrease in expression of several nuclear envelop-associated components (Lamin B1, Lamin A/C, SUN1, Nesprin-3, Plectin) compared to controls. Furthermore, ChIP-qPCR assay showed enrichment of p63 on Sun1, Syne3 and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks H3K27me3, H3K9me3 and heterochromatin protein 1- alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription towards the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression programme in the epidermis.

Osteoarthritis (OA) ist eine chronische Erkrankung der Gelenke des menschlichen Körpers, insbesondere des Kniegelenkes. Sie ist durch entzündliche und degenerative Prozesse gekennzeichnet, die Patienten in ihrer Beweglichkeit stark einschränkt. In der komplexen Pathophysiologie kommt es unter anderem zu zellmorphologischen Veränderungen der knorpelbildenden Zellen, den Chondrozyten, und zu destruktiven Veränderungen der Knorpelmatrix. Bisherige therapeutische Ansätze bestehen in meist in einer rein symptomatischen Therapie durch Schmerzmittel sowie der operativen endoprothetischen Versorgung als Ultima Ratio. Eine kurative Therapie ist bisher nicht möglich. Einen Ansatz für eine kurative Therapie könnte eine Subpopulation der Zellen des Knorpelgewebes bieten. Chondrogene Progenitor Zellen (CPCs) stellen als Vorläuferzellen der Chondrozyten, gesteuert durch das prochondrogene Gen SOX9 und das proosteogene Gen RUNX2, einen möglichen regenerativen Ansatz in der Behandlung dar. Eine Rolle in diesem Prozess könnten die Wachstumsfaktoren TGF- β3 und EGF sowie das Matrixmolekül Biglycan darstellen. In dieser Arbeit konnte gezeigt werden, dass diese Wachstumsfaktoren, deren Rezeptoren und das Matrixmolekül Biglycan im osteoarthrischen Knorpel eine Rolle spielen. Insbesondere konnte in vitro gezeigt werden, dass CPCs unter dem Einfluss dieser Moleküle zu einer vermehrten SOX9 und verminderten RUNX2-Expression angeregt werden. Unter der Hypothese, dass sich CPCs auf diese Art zu Chondrozyten differenzieren lassen und so den Knorpel wiederherstellen, könnten diese Moleküle einen möglichen Baustein einer zukünftigen Therapie der OA darstellen.

Qualificar as primeiras unidades de Sangue de Cordão Umbilical e Placentá- rio (SCUP) criopreservadas no Banco de Sangue Umbilical e placentário da Fundação de Hematologia e Hemoterapia de Pernambuco (BSCUP/HEMOPE), no período de dezembro de 2014 a junho de 2017. Justificativa: O sangue do cordão é rico em células progenitoras hematopoéticas (CPH), utilizado para transplante e tratamento de patologias benignas e malignas. Avaliar as primeiras unidades coletadas no BSCUP/ HEMOPE será de grande valia para sua caracterização, planejamento de ações de melhoria do processo, contribuindo para o aumento da eficácia dos transplantes. Métodos: Foram triadas gestantes sem histórico de diagnóstico para doenças transmissíveis pelo sangue, com gestação terminada em recém-nascido (RN) vivo; caracterizadas através das variáveis sociodemográficas: idade, idade gestacional, antecedentes obstétricos e cor da pele dos RN foram caracterizados pelas variáveis explicativas: Gênero e Peso; as amostras das mães foram coletadas no dia do parto ou em até 48h após. O tempo entre o término da coleta e o início da criopreservação da unidade das CPH não excedeu 48 horas. As unidades de SCUP foram avaliadas quanto ao volume, total de células nucleadas (TCN), quantificação de células CD 34+, contagem de eritroblastos viabilidade celular e contaminação microbiológica. Neste estudo foi avaliada a variável \"unidades adequadas ao uso em relação ao peso do receptor, tomando com o base o TCN e o peso de um provável receptor, assim categorizados: unidades <12,5 x10e8 para peso 12,5 para peso >50kg; Resultados: Volume Inicial (ml):(min.50,0; max.166,80); Volume final(mL): (min.19,07;max.21,75); TCN pré (x108): (min.6,0;max.27,80);TCN pós(x108): (min.5,0; max.22,6); Recuperação Celular (%): (min.0,67; max.39,50); CD34+(x106):(min.0,67;max.39,50);Viabilidade(%): (min.71,35max.100); Conclusão: Das 113 unidades armazenadas, 89 (78,76%) atendem a receptores < 50kg (crianças) e 24 (21,24%) atendem a receptores >50k (adultos). O inventário apresentou resultados em conformidades com o especificado na legislação vigente, portanto, qualificadas para atender à demanda transfusional de transplantes de CPH. / To qualify the first units of cryopreserved Umbilical Cord and Placental Blood (SCUP) in the Umbilical and Placental Blood Bank of the Hematology and Hemotherapy Foundation of the State of Pernambuco (BSCUP / HEMOPE), from December 2014 to June 2017 Rationale: Cord blood is rich in hematopoietic progenitor cells (HPC), used for transplantation and treatment of benign and malignant pathologies. Evaluating the first units collected in the BSCUP / HEMOPE will be of great value for its characterization, planning of actions to improve the process, contributing to increase the efficiency of transplants. Methods: Pregnant women with no history of diagnosis for blood-borne diseases were screened, with gestation terminated in live newborn (NB); characterized by the sociodemographic variables: age, gestational age, obstetric history and skin color, NB were characterized by the explanatory variables: Gender and Weight; the samples of the mothers were collected on the day of delivery or within 48 hours after delivery. The time between the end of the collection and the beginning of the cryopreservation of the MHC unit did not exceed 48 hours. SCUP units were evaluated for volume, total nucleated cells (TNC), CD34 + cell count, cell viability erythroblast counts, and microbiological contamination. In this study, the variable \"units suitable for use in relation to the weight of the receptor\" was evaluated, taking as base the TNC and the weight of a probable receiver, as follows: units <12.5 x10e8 for weight 12 , 5 for weight> 50 kg; Results: Initial volume (ml): (min.50.0, max.166.80); Final volume (mL): (min.19.07, max.21.75); TCN pre (x108): (min.6,0; max.27,80); TCN post (x108): (min.5,0; max.22,6); Cellular Recovery (%): (min.0.67, max.39,50); CD34 + (x106): (min.0.67, max.39.50); Viability (%): (min.71,35max.100); : Of the 113 stored units, 89 (78.76%) attend to receptors <50kg (children) and 24 (21.24%) attend receptors> 50k (adults). Conclusion: The inventory presented results in compliance with that specified in current legislation, therefore, qualified to meet the transfusional demand for HPC transplants.

No description available.

Human induced pluripotent stem cells (hiPSCs) have two main properties: pluripotency and self-renewal. Physical cues presented by biomaterial scaffolds can stimulate differentiation of hiPSCs to neurons. In this work, we develop and analyze a mathematical model of aggregate growth and neural differentiation on melt electrospun biomaterial scaffolds. An ordinary differential equation model of population size of each cell state (stem, progenitor, differentiated) was developed based on experimental results and previous literature. Analysis and numerical simulations of the model successfully capture many of the dynamics observed experimentally. Analysis of the model gives optimal parameter sets, that correspond to experimental procedures, to maximize particular populations. The model indicates that a physiologic oxygen level (~5%) increases population sizes compared to atmospheric oxygen levels (~21%). Model analysis also indicates that the optimal scaffold porosity for maximizing aggregate size is approximately 63%. This model allows for the use of mathematical analysis and numerical simulations to determine the key factors controlling cell behavior when seeded on melt electrospun scaffolds. / Graduate

The role of Notch in cell fate determination and lineage restriction in the bone marrow (BM) is controversial in the field. Recent studies have convincingly shown that Notch is dispensable for haematopoietic stem cell (HSC) regulation in adult haematopoiesis (Maillard et al., 2008). In contrast, Notch signaling has been proposed to be of importance in the regulation of BM megakaryocyte progenitor differentiation, based on dominant negative genetic approaches, identifying a potentially distinct role for Notch in adult BM haematopoiesis (Mercher et al., 2008). Here, I found that by selectively ablating the gene coding the transcription factor recombination signal-binding protein J kappa (RBP-Jk), to which all canonical Notch signaling converges, canonical Notch signaling does not mediate HSC maintenance, neither in steady state nor in conditions of stress. Furthermore, I propose, in contrast with previous studies (Mercher et al., 2008), that canonical Notch signaling plays no role in myeloerythropoiesis cell lineage commitment in the BM. My data also show that key Notch target genes are suppressed by RBP-Jk, as their expression is unaffected in Notch1-deficient BM progenitors, while target genes are upregulated in Rbp-Jk-deleted megakaryocyte and erythroid progenitors. This establishes for the first time in mammalian cells in vivo, that Notch target genes are kept in a suppressed state by RBP-Jk, potentially restricting T cell commitment to the thymus and not to the BM, at the expense of myeloerythropoiesis. Notch signaling and GATA3 are two master regulators in T cell commitment (Han et al., 2002; Ho et al., 2009; Pui et al., 1999; Radtke et al., 1999; Zhu et al., 2004). However, although very well established as being involved in the thymic stages of T cell restriction, there is little evidence of Notch and GATA3 being involved in the migration of a thymus settling progenitor (TSP) from the BM to the thymus or in the establishment of the earliest thymic progenitor (ETP) in the thymus. From this thesis work, I conclude that Notch signaling is essential for the emergence of ETPs in the thymus in a NOTCH1-independent manner. Moreover, I demonstrate, as supported by a very recent published study (Hosoya et al., 2009), that GATA3 is important for the development of the earliest T cell progenitor. GATA1 and GATA2 mediate haematopoietic stem cell maintenance in the BM. GATA1 is required for erythropoiesis, megakaryocytes and eosinophils while GATA2 is important for the proliferation and survival of HSCs. In contrast, a role for GATA3 in the BM has never been established. By using a Gata3-conditional knockout mouse model, I demonstrate that GATA3 is dispensable for HSC maintenance in steady state and following active haematopoietic regeneration as well as for HSC self-renewal in the BM.

Background: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1(-/-)mice and Pim1(-/-)2(-/-)3(-/-) triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 (-/-) and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit S-35 methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Les progéniteurs endothéliaux semblent offrir de nouvelles perspectives dans le traitement des pathologies ischémiques. Nos travaux portent sur l'étude des effets d'une transplantation d'une sous population homogène de progéniteurs endothéliaux dits tardifs, les ECFCs, sur un modèle d'occlusion de l'artère cérébrale moyenne (MCAO) transitoire chez le rat. 4x106 ECFCs cultivés à partir du sang de cordon humain ou 1ml de PBS ont été injectés en intraveineuse (IV) 24h après une MCAO d'une durée d'une heure chez les rats appartenant respectivement au groupe greffé et au groupe contrôle. On a pu mettre en évidence le passage des cellules greffées dans l'hémisphère cérébral ischémié par radiomarquage à l'oxinate d'indium 111 (111In) et marquage fluorescent au CM-Dil des ECFCs avant transplantation. Ceci a été confirmé par la visualisation d'ECFCs d'origine humaine en périphérie de la zone infarcie par marquages immunohistochimiques au MAB1281 et CD31. La transplantation d'ECFCs a augmenté significativement le taux de survie et a amélioré la récupération fonctionnelle des animaux. L'effet bénéfique observé est associé à une réduction du nombre de cellules apoptotiques ainsi qu'une augmentation de la densité capillaire et de la neurogenèse en périphérie de la zone lésée. Ces effets semblent corrélés à une surexpression en zone de pénombre de VEGF et IGF1 aux propriétés pro-angiogéniques et neurotrophiques, et à une diminution de l'expression d'un facteur pro-apoptotique proBDNF. De plus, nous avons montré que les ECFCs sont capables de sécréter des cytokines pro-angiogéniques. / Endothelial progenitor cells (EPCs) seem to be a promising option to treat patients with ischemic diseases. Here, we investigated the effects of late EPCs or Endothelial Colony-Forming Cells (ECFCs), a recently defined homogeneous subtype of EPCs, in a rat model of transient middle cerebral artery occlusion (MCAO). Either vehicle or 4.106 ECFCs, isolated from human cord blood, were intravenously injected 24h after 1 hour of MCAO in rats assigned to control and transplanted groups respectively. 111In-oxine-labeled ECFCs specifically homed to ischemic hemisphere and CM-Dil prelabeled ECFCs preferentially settled in the inner boundary of the core area of transplanted animals. The presence of human cells in rat brain sections was detected by immunohistochemical staining (MAB1281, CD31). We demonstrated that ECFCs injected 24h after MCAO improved functional recovery and survival rate. Beneficial effect was associated with an increase in growth factors expression in homogenates from ischemic area (VEGF, IGF-1, proBDNF) and may be related to the secretion by ECFCs of soluble factors that could affect apoptosis, vascular growth and neurogenesis. These findings raise perspectives for the use of ECFCs as a well-characterized cell therapy product for optimal therapeutic outcome after stroke.

Pour maintenir l'architecture du tissue, les cellules épithéliales se divisent de manière planaire, perpendiculaire à leur axe principal de polarité. Du fait que le centrosome retrouve sa localisation apicale à l'interphase l'orientation du fuseau mitotique est réinitialisée à chaque cycle cellulaire. Nous utilisons de l'imagerie live en trois dimensions de centrosome marqués en GFP pour investiguer la dynamique de l'orientation du fuseau mitotique des cellules neuroépithéliales de l'embryon de poulet. Le fuseau mitotique présente des mouvements stéréotypiques pendant la métaphase, avec dans un premier temps une phase active de d'orientation planaire suivie par une phase de maintenance planaire jusqu'à l'anaphase. Nous décrivons la localisation des protéines NuMA et LGN formant un anneau au niveau du cortex latéral cellulaire au moment de l'orientation du fuseau. Enfin, nous montrons que le complexe protéique formé par LGN, NuMA et par la sous unité Gai localisé au cortex est nécessaire pour les mouvements du fuseau et pour réguler la dynamique de l'orientation du fuseau. La localisation restreinte de LGN et NuMA en anneau cortical est instructive pour l'alignement planaire du fuseau mitotique et est également requise pour sa maintenance planaire. / To maintain tissue architecture, epithelial cells divide in a planar fashion, perpendicular to their main polarity axis. As the centrosome resumes an apical localization in interphase, planar spindle orientation is reset at each cell cycle. We used three-dimensional live imaging of GFP-labeled centrosomes to investigate the dynamics of spindle orientation in chick neuroepithelial cells. The mitotic spindle displays stereotypic movements during metaphase, with an active phase of planar orientation and a subsequent phase of planar maintenance before anaphase. We describe the localization of the NuMA and LGN proteins in a belt at the lateral cell cortex during spindle orientation. Finally, we show that the complex formed of LGN, NuMA, and of cortically located Gái subunits is necessary for spindle movements and regulates the dynamics of spindle orientation. The restricted localization of LGN and NuMA in the lateral belt is instructive for the planar alignment of the mitotic spindle, and required for its planar maintenance.