Functional analysis of different haematopoietic stem cell subsets

RÉSUMÉ : Elucider les bases moléculaires et cellulaires du fonctionnement des cellules souches s'avère crucial dans la compréhension de l'organisation cellulaire au sein des tissus et des organes ainsi que pour le développement de nouvelles stratégies thérapeutiques en médecine régénérative et en oncologie. Les cellules souches adultes les mieux connues sont celles responsables de l'hématopoïèse, les cellules souches hématopoïétiques (CSH). Durant ces dernières années, la recherche a porté une attention particulière à l'isolation prospective de CSH dérivées de la moelle osseuse de souris en utilisant des marqueurs de surface cellulaire ainsi que des propriétés fonctionnelles alléguées. Par la suite, la capacité fonctionnelle des CSH a été vérifiée classiquement par leur transplantation intraveineuse dans des souris réceptrices conditionnées et par l'analyse de leur aptitude à reconstituer le système hématopoïétique à long terme. Des études récentes suggèrent que la transplantation des cellules directement dans la moelle osseuse pourrait non seulement aboutir à une prise de greffe plus rapide et plus efficace, mais pourrait même aider à l'identification de cellules qui ont certes des propriétés intrinsèques de CSH, mais qui n'ont pas la capacité de trouver leur niche au sein de la moelle osseuse et ont donc échoué dans les analyses classiques de reconstitution. Dans cette étude, nous comparons à deux niveaux la fonction de différents sous-groupes de cellules souches de la moelle osseuse, définis par leur phénotype de surface cellulaire. Premièrement, nous étudions leur capacité à reconstituer des souris létalement irradiées après injection soit intraveineuse soit intrafémorale. Deuxièmement, par analyse cytométrique de flux à 8 couleurs, nous comparons leur activité relative de « side population » (SP) par exclusion du colorant fluorescent Hoechst 33342. Nos résultats préliminaires renforcent en effet l'idée que la transplantation intrafémorale aboutit à une greffe plus rapide et plus efficace. Par contre, en utilisant cette approche, nous n'arrivons pas à identifier des cellules capables de prendre greffe spécifiquement quand elles sont injectées en intrafémorale. Finalement, bien qu'une confirmation in vivo soit encore nécessaire, nous suggérons sur la base de nos analyses cytométriques de flux, que les cellules SP Sca1t~és éie~~ CD48t~és bas sont très enrichies en CSH. Ceci permettrait l'isolation ex vivo de CSH de la moelle osseuse de souris par une stratégie à la fois nouvelle et simple. SUMMARY : Elucidating the molecular and cellular bases of stem cell function is crucial for the understanding of cellular organisation within tissues and organs as well as for the development of new therapeutic strategies in regenerative medicine and oncology. The best-known adult stem cells are those responsible for haematopoiesis, the haematopoietic stem cells (HSCs). In recent years, much effort has been put into the prospective isolation of mouse bone marrow (BM)-derived HSCs using cell-surface markers and alleged functional properties. Upon isolation, the functional capacity of putative HSCs has been classically assessed by intravenous transplantation into conditioned recipient mice and analysis of their ability to reconstitute the haematopoietic system at long-term. It has recently been suggested that transplanting the cells directly into the BM might not only result in more rapid and more effective engraftment, but even help to identify cells that have intrinsic HSC properties but lack the ability to home to their BM niche and have thus failed to succeed in classical reconstitution assays. In this study, we compare the function of different BM cell subsets, as defined by their cell surface phenotype, on two levels. Firstly, we assess their ability to reconstitute lethally irradiated mice, when injected either intravenously or intrafemorally. Secondly, using 8-colour flow cytometric analysis, we compare their relative side population (SP) activity by exclusion of the fluorescent dye Hoechst 33342. Our preliminary results indeed reinforce the idea that intrafemoral transplantation results in faster and more effective engraftment, however, using this approach, we are unable to identify cells that are capable of engrafting specifically when injected intrafemorally. Finally, although in vivo confirmation is still required, we propose, based on the results of our flow cytometric analyses, that SP Scat Very h'9h CD48Very'°W cells should be highly enriched for HSCs. This would allow for a simple new strategy for the isolation of mouse BM HSCs ex vivo.

Concise reviews: in vitro-produced pancreas organogenesis models in three dimensions: self-organization from few stem cells or progenitors.

Three-dimensional models of organ biogenesis have recently flourished. They promote a balance between stem/progenitor cell expansion and differentiation without the constraints of flat tissue culture vessels, allowing for autonomous self-organization of cells. Such models allow the formation of miniature organs in a dish and are emerging for the pancreas, starting from embryonic progenitors and adult cells. This review focuses on the currently available systems and how these allow new types of questions to be addressed. We discuss the expected advancements including their potential to study human pancreas development and function as well as to develop diabetes models and therapeutic cells.

Expression and role of BORIS/CTCFL in human cancer stem cells

Le cancer est défini comme la croissance incontrôlée des cellules dans le corps. Il est responsable de 20 % des décès en Europe. Plusieurs expériences montrent que les tumeurs sont issues et se développent grâce à un petit nombre de cellules, que l'on appelle cellules souches cancéreuses (CSC). Ces CSC sont également responsables de l'apparition de métastases et de la résistance aux médicaments anticancéreux. De ce fait, l'identification des gènes qui contribuent aux propriétés de ces CSC (comme la survie des tumeurs, les métastases et la résistance aux médicaments) est nécessaire pour mieux comprendre la biologie des cancers et d'améliorer la qualité des soins des patients avec un cancer. A ce jour, de nombreux marqueurs ont été proposés ainsi que de nouvelles thérapies ciblées contre les CSC. Toutefois, et malgré les énormes efforts de la recherche dans ce domaine, la quasi-totalité des marqueurs de CSC connus à ce jour sont aussi exprimés dans les cellules saines. Ce projet de recherche visait à trouver un nouveau candidat spécifique des CSC. Le gène BORIS (pour Brother of Regulator of Imprinted Sites), nommé aussi CTCFL (CTCF-like), semble avoir certaines caractéristiques de CSC et pourrait donc devenir une cible prometteuse pour le traitement du cancer. BORIS/CTCFL est une protéine nucléaire qui se lie à l'ADN, qui est exprimée dans les tissus normaux uniquement dans les cellules germinales et qui est réactivée dans un grand nombre de tumeurs. BORIS est impliqué dans la reprogrammation épigénétique au cours du développement et dans la tumorigenèse. En outre, des études récentes ont montré une association entre l'expression de BORIS et un mauvais pronostic chez des patients atteints de différents types de cancers. Nous avons développé une nouvelle technologie basée sur les Molecular Beacon pour cibler l'ARNm de BORIS et cela dans les cellules vivantes. Grâce à ce système expérimental, nous avons montré que seule une toute petite sous-population (0,02 à 5%) de cellules tumorales exprimait fortement BORIS. Les cellules exprimant BORIS ont pu être isolées et elles présentaient les caractéristiques de CSC, telles qu'une forte expression de hTERT et des gènes spécifiques des cellules souches (NANOG, SOX2 et OCT4). En outre, une expression élevée de BORIS a été mise en évidence dans des populations enrichies en CSC ('side population' et sphères). Ces résultats suggèrent que BORIS pourrait devenir un nouveau et important marqueur de CSC. Dans des études fonctionnelles sur des cellules de cancer du côlon et du sein, nous avons montré que le blocage de l'expression de BORIS altère largement la capacité de ces cellules à former des sphères, démontrant ainsi un rôle essentiel de BORIS dans l'auto- renouvellement des tumeurs. Nos expériences montrent aussi que BORIS est un facteur important qui régule l'expression de gènes jouant un rôle clé dans le développement et la progression tumorale, tels le gène hTERT et ceux impliqués dans les cellules souches, les CSC et la transition épithélio-mésenchymateuse (EMT). BORIS pourrait affecter la régulation de la transcription de ces gènes par des modifications épigénétiques et de manière différente en fonction du type cellulaire. En résumé, nos résultats fournissent la preuve que BORIS peut être classé comme un gène marqueur de cellules souches cancéreuse et révèlent un nouveau mécanisme dans lequel BORIS jouerait un rôle important dans la carcinogénèse. Cette étude ouvre de nouvelles voies pour mieux comprendre la biologie de la progression tumorale et offre la possibilité de développement de nouvelles thérapies anti-tumorales et anti-CSC avec BORIS comme molécule cible. - Cancer is defined as the uncontrolled growth of cells in the body. It causes 20% of deaths in the European region. Current evidences suggest that tumors originate and are maintained thanks to a small subset of cells, named cancer stems cells (CSCs). These CSCs are also responsible for the appearance of metastasis and therapeutic resistance. Consequently, the identification of genes that contribute to the CSC properties (tumor survival, metastasis and therapeutic resistance) is necessary to better understand the biology of malignant diseases and to improve care management. To date, numerous markers have been proposed to use as new CSC- targeted therapies. Despite the enormous efforts in research, almost all of the known CSCs markers are also expressed in normal cells. This project aimed to find a new CSC-specific candidate. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA binding protein involves in epigenetic reprogramming in normal development and in tumorigenesis. Recent studies have shown an association of BORIS expression with a poor prognosis in different types of cancer patients. Therefore, BORIS seems to have the same characteristics of CSCs markers and it could be a promising target for cancer therapy. BORIS is normally expressed only in germinal cells and it is re-expressed in a wide variety of tumors. We developed a new molecular beacon-based technology to target BORIS mRNA expressing cells. Using this system, we showed that the BORIS expressing cells are only a small subpopulation (0.02-5%) of tumor cells. The isolated BORIS expressing cells exhibited the characteristics of CSCs, with high expression of hTERT and stem cell genes (NANOG, SOX2 and OCT4). Furthermore, high BORIS expression was observed in the CSC-enriched populations (side population and spheres). These results suggest that BORIS might be a novel and powerful CSCs marker. In functional studies, we observed that BORIS knockdown significantly impairs the capacity to form spheres in colon and breast cancer cells, thus demonstrating a critical role of BORIS in the self-renewal of tumors. The results showed in the functional analysis indicate that BORIS is an important factor that regulates the expression of key-target genes for tumor development and progression, such as hTERT, stem cells, CSCs markers and EMT (epithelial mesenchymal transition)-related marker genes. BORIS could affect the transcriptional regulation of these genes by epigenetic modification and in a cell type dependent manner. In summary, our results support the evidence that BORIS can be classified as a cancer stem cell marker gene and reveal a novel mechanism in which BORIS would play a critical role in tumorigenesis. This study opens new prospective to understand the biology of tumor development and provides opportunities for potential anti-tumor drugs.

The role of Bmi1 in CNS development and in retinal degeneration

SUMMARY : The present work addresses several aspects of cell cycle regulation, cell fate specification and cell death in the central nervous system (CNS), specifically the cortex and the retina. More precisely, we investigated the role of Bmi1, a polycomb family gene required for stem cell proliferation and self-renewal, in the development of the cerebral cortex, as well as in the genesis of the retina. These data, together with studies published during the last two decades concerning cell cycle re-activation in apoptotic neurons in the CNS, raised the question of a possible link between regulation of the cell cycle during development and during retinal degeneration. 1. The effects of Bmi1 loss in the cerebral cortex : Consistently with our and others' observations on failure of Bmi9-/- stem cells to proliferate and self-renew in vitro, the Bmi9-/- cerebral cortex presented slight defects in proliferation in stem/progenitor cells compartments in vivo. This was in accordance with the pattern of Bmi1 expression in the developing forebrain. The modest proliferation defects, compared to the drastic consequences of Bmi9 loss in vitro, suggest that cell-extrinsic mechanisms may partially compensate for Bmi1 deletion in vivo during cortical histogenesis. Nevertheless, we observed a decreased proliferating activity in neurogenic regions of the adult telencephalon, more precisely in the subventricular zone, showing that Bmi1 controls neural stem/progenitor proliferation during adulthood in vivo. Our data also highlight an increased production of astrocytes at birth, and a generalized gliosis in the adult Bmi9-/- brain. Importantly, glial progenitors and astrocytes retained the ability to proliferate in the absence of Bmi1. 2. The effects of Bmi1 loss in the retina : The pattern of expression of Bmi1 during development and in the adult retina suggests a role for Bmi1 in cell fate specification and differentiation rather than in proliferation. While the layering and the global structure of the retina appear normal in Bmi1 /adult mice, immunohistochemìcal analysis revealed defects in the three major classes of retinal interneurons, namely: horizontal, bipolar and amacrine cells. Electroretinogram recordings in Bmi9-/- mice are coherent with the defects observed at the histological level, with a reduced b-wave and low-profile oscillatory potentials. These results show that Bmi1 controls not only proliferation, but also cell type generation, as previously observed in the cerebellum. 3. Cell cycle events and related neuroprotective strategies in retinal degeneration : In several neurodegenerative disorders, neurons re-express cell cycle proteins such as cyclin dependent kinases (Cdks) prior to apoptosis. Here, we show for the first time that this is also the case during retinal degeneration. Rd1 mice carry a recessive defect (Pdeóbrd/rd) that causes retinal degeneration and serves as a model of retinitis pigmentosa. We found that photoreceptors express Cdk4 and Cdk2, and undergo DNA synthesis prior to cell death. To interfere with the reactivation of Cdk-related pathways, we deleted E2fs or Brni1, which normally allow cell cycle progression. While deleting E2f1 (downstream of Cdk4/6) in Rd1 mice provides only temporary protection, knocking out Bmi1 (upstream of Cdks) leads to an extensive neuroprotective effect, independent of p16ink4a or p19arf, two tumor suppressors regulated by Bmi1. Analysis of Cdks and the DNA repair-related protein Ligase IV showed that Bmi1 acts downstream of DNA repair events and upstream of Cdks in this neurodegenerative mechanism. Expression of Cdks during an acute model of retinal degeneration, light damage-induced photoreceptor death, points to a role for Bmi1 and cell cycle proteins in retinal degeneration. Considering the similarity with the cell cycle-related apoptotic pathway observed in other neurodegenerative diseases, Bmi1 is a possible general target to prevent or delay neuronal death. RESUME : Ce travail aborde plusieurs aspects de la régulation du cycle cellulaire, de la spécification du devenir des cellules et de la mort cellulaire dans le système nerveux centrale (SNC), plus particulièrement dans le cortex cérébral et dans la rétine. Nous nous sommes intéressés au gène Bmi1, appartenant à la famille polycomb et nécessaire à la prolifération et au renouvellement des cellules souches. Nous avons visé à disséquer son rôle dans le développement du cortex et de la rétine. Ces données, ainsi qu'une série de travaux publiés au cours des deux dernières décennies concernant la réactivation du cycle cellulaire dans les neurones en voie d'apoptose dans le SNC, nous ont ensuite poussé à chercher un lien entre la régulation du cycle cellulaire pendant le développement et au cours de la dégénérescence rétinienne. 1. Les effets de l'inactivation de Bmi1 dans le cortex cérébral : En accord avec l'incapacité des cellules souches neurales in vitro à proliférer et à se renouveler en absence de Bmi1, le cortex cérébral des souris Bmi1-/- présente de légers défauts de prolifération dans les compartiments contenant les cellules souches neurales. Ceci est en accord avec le profil d'expression de Bmi1 dans le télencéphale. Les conséquences de la délétion de Bmi1 sont toutefois nettement moins prononcées in vivo qu'in vitro ; cette différence suggère l'existence de mécanismes pouvant partiellement compenser l'absence de Bmi1 pendant la corticogenèse. Néanmoins, l'observation d'une réduction de la prolifération dans la zone sous-ventriculaire, la zone majeure de neurogenèse dans le télencéphale adulte, montre que Bmi1 contrôle la prolifération des cellules souche/progénitrices neurales chez la souris adulte. Nos résultats démontrent par ailleurs une augmentation de la production d'astrocytes à la naissance ainsi qu'une gliose généralisée à l'état adulte chez les souris Bmi1-/-. Les progéniteurs gliaux et les astrocytes conservent donc leur capacité à proliférer en absence de Bmi1. 2. Les effets de l'inactivation de Bmi1 dans la rétine : Le profil d'expression de Bmi1 pendant fe développement ainsi que dans la rétine adulte suggère un rôle de Bmi1 dans la spécification de certains types cellulaires et dans la différentiation plutôt que dans la prolifération. Alors que la structure et la lamination de la rétine semblent normales chez les souris Bmi1-/-, l'analyse par immunohistochimie amis en évidence des défauts au niveau des trois classes d'interneurones rétiniens (les cellules horizontales, bipolaires et amacrines). Les électrorétinogrammes des souris Bmi1-/- sont cohérents avec les défauts observés au niveau histologique et montrent une réduction de l'onde « b » et des potentiels oscillatoires. Ces résultats montrent que Bmi1 contrôle la génération de certaines sous-populations de neurones, comme démontré auparavant au niveau de cervelet. 3. Réactivation du cycle cellulaire et stratégies théraoeutiaues dans les dégénérescences rétiniennes : Dans plusieurs maladies neurodégénératives, les neurones ré-expriment des protéines du cycle cellulaire telles que les kinases cycline-dépendantes (Cdk) avant d'entrer en apoptose. Nous avons démontré que c'est aussi le cas dans les dégénérescences rétiniennes. Les souris Rd1 portent une mutation récessive (Pde6brd/rd) qui induit une dégénérescence de la rétine et sont utilisées comme modèle animal de rétinite pigmentaire. Nous avons observé que les photorécepteurs expriment Cdk4 et Cdk2, et entament une synthèse d'ADN avant de mourir par apoptose. Pour interférer avec la réactivation les mécanismes Cdk-dépendants, nous avons inactivé les gènes E2f et Bmi1, qui permettent normalement la progression du cycle cellulaire. Nous avons mis en évidence que la délétion de E2f1 (en aval de Cdk4/6) dans les souris Rd1 permet une protection transitoire des photorécepteurs. Toutefois, l'inactivation de Bmi1 (en amont des Cdk) est corrélée à une neuroprotection bien plus durable et ceci indépendamment de p16ink4a et p19arf, deux suppresseurs de tumeurs normalement régulés par Bmi1. L'analyse des Cdk et de la ligase IV (une protéine impliquée dans les mécanismes de réparation de l'ADN) a montré que Bmi1 agit en aval des événements de réparation de l'ADN et en amont des Cdk dans la cascade apoptotique dans les photorécepteurs des souris Rd1. Nous avons également observé la présence de Cdk dans un modèle aigu de dégénérescence rétinienne induit par une exposition des animaux à des niveaux toxiques de lumière. Nos résultats suggèrent donc un rôle général de Bmi1 et des protéines du cycle cellulaire dans les dégénérescences de la rétine. Si l'on considère la similarité avec les événements de réactivation du cycle cellulaire observés dans d'autres maladies neurodégénératives, Bmi1 pourrait être une cible thérapeutique générale pour prévenir la mort neuronale.

Homeostasis limits the development of mature CD8+ but not CD4+ thymocytes.

The involvement of a variety of clonal selection processes during the development of T lymphocytes in the thymus has been well established. Less information, however, is available on how homeostatic mechanisms may regulate the generation and maturation of thymocytes. To investigate this question, mixed radiation bone marrow chimeras were established in which wild-type T cell precursors capable of full maturation were diluted with precursors deficient in maturation potential because of targeted mutations of the RAG1 or TCR-alpha genes. In chimeras in which the majority of thymocytes are blocked at the CD4- CD8- CD25+ stage (RAG1 deficient), and only a small proportion of T cell precursors are of wild-type origin, we observed no difference in the maturation of wild-type CD4- CD8- CD25+ cells to the CD4+ CD8+ stage as compared with control chimeras. Therefore, the number of cell divisions occurring during this transition is fixed and not subject to homeostatic regulation. In contrast, in mixed chimeras in which the majority of thymocytes are blocked at the CD4+ CD8+ stage (TCR-alpha deficient), an increased efficiency of development of wild-type mature CD8+ cells was observed. Surprisingly, the rate of generation of mature CD4+ thymocytes was not affected in these chimeras. Thus, the number of selectable CD8 lineage thymocytes apparently saturates the selection mechanism in normal mice while the development of CD4 lineage cells seems to be limited only by the expression of a suitable TCR. These data may open the way to the identification of homeostatic mechanisms regulating thymic output and CD4/CD8 lineage commitment, and the development of means to modulate it.

Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells.

Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1(-/)- BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1(-/)- T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors.

Lymphatic vascular morphogenesis in development, physiology, and disease.

The lymphatic vasculature constitutes a highly specialized part of the vascular system that is essential for the maintenance of interstitial fluid balance, uptake of dietary fat, and immune response. Recently, there has been an increased awareness of the importance of lymphatic vessels in many common pathological conditions, such as tumor cell dissemination and chronic inflammation. Studies of embryonic development and genetically engineered animal models coupled with the discovery of mutations underlying human lymphedema syndromes have contributed to our understanding of mechanisms regulating normal and pathological lymphatic morphogenesis. It is now crucial to use this knowledge for the development of novel therapies for human diseases.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells.

Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.

Harnessing epithelial stem cell potency

Increase in potency of adult stem/progenitor cells holds great expectations for regenerative medicine; reprogramming is achieved by manipulating the genome or indirectly by manipulating the microenvironment. However, the genetic approach, which can result in lineage conversion up to ground pluripotent embryonic state, will certainly face strict regulatory constraints and consequently translation to the clinic may be difficult. Manipulating stem cell fate without altering the genome of adult stem cells is a promising alternative. My laboratory has demonstrated that non hairy squamous epithelia e.g. the cornea, the oral cavity, the oesophagus, the vagina, contain clonogenic stem cells that can respond to skin morphogenetic signals and form epidermis, cycling hair follicles and sebaceous glands. This capacity is maintained in serial transplantation, crosses primary germ line boundaries and is intrinsic to the stem cells, as cells which have never been exposed to cell culture behave in a similar fashion. Even more surprising, the thymus contains a population of clonogenic epithelial cells of endodermal origin that maintain a thymic identity in culture and have the capacity to incorporate into a thymic network, but can acquire the functionality of bona fide multipotent stem cells of the skin when exposed to proper developmental signals. Thymic epithelial cells exposed to a skin microenvironment exhibit a down-regulation or silencing of transcription factors important for thymic function. Hence, it is possible to reveal unsuspected potency and even to robustly reprogram stem cells by solely manipulating the microenvironment.

Genetic and hormonal regulation of cambial development.

The stems and roots of most dicot plants increase in diameter by radial growth, due to the activity of secondary meristems. Two types of meristems function in secondary plant body formation: the vascular cambium, which gives rise to secondary xylem and phloem, and the cork cambium, which produces a bark layer that replaces the epidermis and protects the plant stem from mechanical damage and pathogens. Cambial development, the initiation and activity of the vascular cambium, leads to an accumulation of wood, the secondary xylem tissue. The thick, cellulose-rich cell walls of wood provide a source of cellulose and have the potential to be used as a raw material for sustainable and renewable energy production. In this review, we will discuss what is known about the mechanisms regulating the cambium and secondary tissue development.

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells.

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.

Group mentoring for women starting a PhD: a "duty free zone" to build professional identity

Despite increasing numbers of women attaining higher level academic degrees, gender disparities remain among higher education and among university faculties. Some have posited that this may stem from inadequate academic identity development of women at the doctoral level. While existing gender differences may stem from multiple and variable origins, mentoring has been proposed as a viable means to promote academic identity development and address these gender gaps. This study used a qualitative, narrative case study design to evaluate "StartingDoc" a structured mentoring program launched among Swiss Universities aimed at promoting networking and academic identity development among female doctoral students. Herein we describe the 9 emergent themes which arose from the small-group mentoring program and suggest that such an approach is both feasible and beneficial for young female academics. Further work is needed to elucidate the most effective strategies for developing and retaining women in academia.

Bone regeneration and stem cells.

?  Introduction ?  Bone fracture healing and healing problems ?  Biomaterial scaffolds and tissue engineering in bone formation -  Bone tissue engineering -  Biomaterial scaffolds -  Synthetic scaffolds -  Micro- and nanostructural properties of scaffolds -  Conclusion ?  Mesenchymal stem cells and osteogenesis -  Bone tissue -  Origin of osteoblasts -  Isolation and characterization of bone marrow derived MSC -  In vitro differentiation of MSC into osteoblast lineage cells -  In vivo differentiation of MSC into bone -  Factors and pathways controlling osteoblast differentiation of hMSC -  Defining the relationship between osteoblast and adipocyte differentiation from MSC -  MSC and sex hormones -  Effect of aging on osteoblastogenesis -  Conclusion ?  Embryonic, foetal and adult stem cells in osteogenesis -  Cell-based therapies for bone -  Specific features of bone cells needed to be advantageous for clinical use -  Development of therapeutic biological agents -  Clinical application concerns -  Conclusion ?  Platelet-rich plasma (PRP), growth factors and osteogenesis -  PRP effects in vitro on the cells involved in bone repair -  PRP effects on osteoblasts -  PRP effects on osteoclasts -  PRP effects on endothelial cells -  PRP effects in vivo on experimental animals -  The clinical use of PRP for bone repair -  Non-union -  Distraction osteogenesis -  Spinal fusion -  Foot and ankle surgery -  Total knee arthroplasty -  Odontostomatology and maxillofacial surgery -  Conclusion ?  Molecular control of osteogenesis -  TGF-β signalling -  FGF signalling -  IGF signalling -  PDGF signalling -  MAPK signalling pathway -  Wnt signalling pathway -  Hedgehog signalling -  Notch signalling -  Ephrin signalling -  Transcription factors regulating osteoblast differentiation -  Conclusion ?  Summary This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Notch regulation of lymphocyte development and function.

Notch proteins regulate a broad spectrum of cell fate decisions and differentiation processes during fetal and postnatal development. Mammals have four Notch receptors that bind five different ligands. The function of Notch signaling during lymphopoiesis and T cell neoplasia, based on gain-of-function and conditional loss-of-function approaches for the Notch1 receptor, indicates Notch1 is essential in T cell lineage commitment. Recent studies have addressed the involvement of other Notch receptors and ligands as well as their downstream targets, demonstrating additional functions of Notch signaling in embryonic hematopoiesis, intrathymic T cell development, B cell development and peripheral T cell function.