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2ND International Conference on Stem Cells and Cancer

ENS Lyon, France, 15-16 November 2016

INV01 Stem cell dynamics during adult hair follicle regeneration and skin wound healing

J BIernaskie 1 1: University of Calgary Self renewing dermal stem cells reside at the base of hair follicles and function to repopulate the mesenchymal compartments with each new regenerative cycle. Here, I will describe our recent work describing the molecular signals that regulate their behavior within this niche, their potential contributions to skin wound healing and the impact of advanced age on dermal stem cell function.

INV02 Integration of cell cycle control and cell fate choice in M-CSF-instructed myeloid lineage commitment of hematopoietic stem cells.

S Sarrazin 1 1: Centre d’Immunologie de Marseille-Luminy (CIML), Marseille, France We have shown previously that under hematopoietic stress conditions of infection high systemic levels of M-CSF can directly instruct myeloid gene expression and differentiation preference of HSC by activation of the myeloid master regulator PU.1 (Mossadegh-Keller et al, Nature, 2013), demonstrating that stem cells are direct targets of lineage instruction by cytokines. Moreover, we have shown earlier that HSC deficient for the transcription factor MafB are more sensible to M-CSF induced PU.1 activation and myeloid lineage commitment (Sarrazin et al, Cell, 2009). It has also been recently shown that cell cycle length in progenitors can control PU.1 protein accumulation and, as a consequence, myeloid lineage commitment (Kueh et al, Science, 2013). Since MafB is known to control cellular proliferation of mature myeloid cells (Tillmanns et al, MCB, 2007; Aziz et al, Science, 2009) we investigated whether MafB levels could control cell cycle entry and thus PU.1 accumulation in M-CSF instructed-HSC.

We monitored cell division and commitment of wt and MafB -/- HSC in response to M-CSF by video-microscopy and analyzed mRNA expression of cell cycle regulators by nanofluidic real time PCR of individual HSC. We observed that in absence of MafB, HSC entered the cycle at a higher rate in response to M-CSF and showed a gene expression signature typical of cycling cells. In vivo, individual transplanted MafB -/- HSC showed rapid activation of PU.1 in a hematopoietic microenvironment of a host with high systemic levels of M-CSF. As in wt HSC, PU.1 activation in MafB deficient HSC was dependent on M-CSF signaling as demonstrated by use of blocking antibody or chemical inhibitor to specifically block M-CSF receptor signaling. Moreover, gene expression profiling of single MafB deficient HSC demonstrated that M-CSF induced a myeloid signature as in wt HSC. Taken together our data indicate that MafB affects cytokine instructed cell fate change in HSC by controlling rate of cell cycle entry. These observations reveal novel mechanisms integrating cell cycle control and lineage choice in HSC. Their manipulation may hold potential for therapeutic modulation of HSC behavior under challenge conditions such as transplantation or infection.

In line with this we could show that M-CSF induced myeloid commitment of HSC is functionally relevant during HSC transplantation where it protects against bacterial and fungal infection during the initial leukopenic phase and thus might have therapeutic potential in the clinic.

INV03 Translational Control of Muscle Stem Cells

C Crist 1 2 1: Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, H3T 1E2, Canada 2: Department of Human Genetics, McGill University, Montreal, Quebec, H3A 1B1, Canada Regeneration of adult tissues depends on somatic stem cells that remain quiescent, yet are primed to enter a differentiation program. The molecular pathways that prevent activation of these cells are not well understood. Using mouse skeletal muscle stem cells as a model, we show that a general repression of translation, mediated by the phosphorylation of translation initiation factor eIF2a at serine 51 (P-eIF2α), is required to maintain the quiescent state. Skeletal muscle stem cells unable to phosphorylate eIF2a exit quiescence, activate the myogenic program and differentiate, but do not self-renew. P-eIF2α ensures in part the robust translational silencing of accumulating mRNAs that is needed to prevent the activation of muscle stem cells. Additionally, P-eIF2α dependent translation of mRNAs regulated by upstream open reading frames (uORFs) contributes to the molecular signature of stemness. Finally, we



show that addition of small molecule inhibitors of eIF2α dephosphorylation to muscle stem cell cultures permits their ex vivo expansion and engraftment into a preclinical mouse model of Duchenne muscular dystrophy. INV04 Secreted niche factors prevent cellular senescence of hematopoietic stem cells

R A J Oostendorp 1 1: Medizinische Klinik, Klinikum rechts der Isar, Munich The niche is required for the lifetime maintenance of hematopoietic stem cells (HSCs). The main function of HSCs is the restoration of the hematopoietic hierarchy after hematopoietic stress, such as inflammatory or toxic insult, or transplantation. Importantly, alterations in niche function collaborate in leukemogenesis and are involved in the diminished HSC function in aging.

In order to identify secreted niche gene products involved in HSC regulation, we evaluated the transcriptome of stromal cell culture models of stress. These studies showed that high expression of Sfrp1, Sfrp2, and Ptn were required for maintaining HSC function. In transcriptomes from co-culture studies, we found a dynamic reciprocal network of interactions between HSCs and stromal cells and identified induction of stromal Ctgf to be important for allowing HSC proliferation.

The deletion of Ctgf, Sfrp1, or Sfrp2 in stromal cell line UG26-1B6, leads to inability to maintain repopulating HSCs in vitro. In respective Sfrp-mutant mice, we found no major alterations in steady-state hematopoeisis. However, the in vivo stress response in these mice is severely impaired and transplantation of wild-type HSCs in Sfrp1-/- or Sfrp2-/-

mice (a model of regenerative stress) shows diminished repopulation of donor HSCs.

To determine the underlying mechanisms of diminished HSC function in niche-mutant environments, we performed clonal cell division studies of HSCs in conditioned media (CM) from stromal cells deficient in Ctgf or Sfrp1. These studies showed that HSCs grow less well in such CMs compared to CM from control stroma. Also, in the clonal cultures in shSfrp1- and shCtgf-CM, HSC showed more differentiation. These effects could be counteracted by adding recombinant SFRP1 or CTGF, respectively. To find out underlying mechanisms of HSC growth arrest, we found that HSCs grown in shSfrp1- and shCtgf-CM show an accumulation of gH2A.X+ heterochromatin foci and acetylated H4K16. In Ctgf-deficient stromal cell media, this is also accompanied by activation of SMAD2/3 and PTEN in HSCs. These markers are frequently found in cellular senescence. Interestingly, in vivo, HSCs from a Sfrp2 deficient environment show similar changes in senescence marker expression after transplantation, 5-fluorouracil treatment or upon aging.

Taken together, our studies support the view that different secreted niche factors are required to maintain cell division behavior and stabilize gH2A.X-dependent DNA stability under conditions of culture stress in vitro and regenerative/ replicative stress in vivo. Lack of the secreted niche factors results in increased DNA damage and heterochromatin formation, which is associated with delayed cell division and HSCs exhaustion. Our studies offer new targets in CTGF and SFRP1/SFRP2 signaling to prevent cellular senescence in vitro (expansion), or, alternatively, reactivate senescence in malignancies in which alterations allowing senescence evasion have occurred.

INV05 AMPKα1 as a regulator of adult stem cell fate choice

R Mounier 1 1: INMG, Université Lyon 1 AMPK functions as a fuel sensor to coordinate the balance between energy consuming vs. producing processes, but also regulating metabolic processes maintaining cell energy homeostasis. AMPK activation induces cell cycle arrest associated with the stabilisation of the tumour suppressor p53, but also promotes a switch from rapid glucose uptake, glycolysis and lactate output to oxidative metabolism. Moreover AMPK activation is downregulated in many tumour cells by the loss of LKB1. Nevertheless, AMPK action(s) on stem cell fate choice has not been investigated. This is of interest concerning the cancer stem cells (CSCs) which, depending on the environment remain dormant, or reactivate and give rise to the most aggressive and metastatic cancer cell populations.

However, CSC phenotype is highly variable and universal robust markers to identify them have not been defined yet. To overcome this issue, we use a very well defined model of adult stem cells: the muscle stem cell (MuSC). Upon muscle injury, MuSCs (quiescent in their niche in normal muscle) become activated, proliferate and choose between



myogenic terminal differentiation and self-renewal to return to quiescence. Interestingly, common signalling pathways finely tune MuSC fate and regulate the pool of CSCs.

In this context, we decipher the role of AMPK in adult stem cell fate and particularly during the choice governing the acquisition of the quiescent state. In vivo, loss-of-function experiments in MuSCs permit us to point out that in absence of AMPKα1, MuSCs differentiate in a lower extend and self-renew much more. Metabolism of MuSCs is explored in vitro analyzing the balance of oxidative/glycolytic use.

Our results support the hypothesis that AMPKα1-/- muscle stem cells preferably use the glycolysis and are not able to respond to the energy demand of the myogenic program, inducing a higher return to quiescence. Finally, we have discovered a new functional target of AMPKα1 in this context that allow us to decipher the complete molecular pathway of AMPKα1 involved in stem cell fate.

To conclude, our work permits to establish a new and crucial role of AMPKα1 in MuSCs fate choice by switching the metabolism during skeletal muscle regeneration, linking for the very first time self renewal and metabolism in this context.

INV06 Nutrient resource management in hematopoietic stem cell differentiation

N Taylor 1 1: Institut de Génétique Moléculaire, Montpellier The self renewal capacity of hematopoietic stem cells (HSCs) is controlled by the cells’ metabolic state but the possibility that nutrient entry and metabolism contribute to the differential commitment of an HSC to a specific lineage fate was not considered until very recently. Our group has shown that fuel availability and utilization condition HSC and progenitor cell fate. Specifically, inhibiting the ASCT2 glutamine transporter or downstream glutaminolysis diverts erythropoietin-signaled human HSCs to a myeloid cell fate. Erythroid specification requires glutamine-dependent de novo nucleotide biosynthesis and shunting glucose through the pentose phosphate pathway, thereby increasing nucleotide synthesis, accelerates erythropoiesis (Oburoglu et al., CSC, 2014). Metabolic reprogramming, such as that occurring in the physiological context of erythropoiesis, is also regarded as a hallmark of cancer. The mTOR pathway integrates cellular energetics and tumor growth and we will present data revealing the importance of this pathway, together with downstream changes in fatty acid oxidation, in HSC commitment to an erythroid versus myeloid lineage fate. These data support our hypothesis that the utilization of nutrient resources governs HSC differentiation at a level beyond simply providing ATP for energy-consuming cellular processes, with metabolic alterations contributing to the dysregulated hematopoiesis that characterizes patients with hematopoietic malignancies.

INV07 Leukemic stem cell interactions with the microenvironment: friend or foe?

D BONNET 1 1: The Francis Crick Institute Acute myeloid leukemia (AML) has long been considered a hematopoietic-cell autonomous disorder in which disease initiation and progression is driven by hematopoietic cell intrinsic genetic events. Recent experimental findings in diverse model systems have challenged this view, implicating different stromal cells of the bone marrow in disease pathogenesis. Thus it is now accepted that leukemic hematopoiesis can turn the BM niche into a “leukemic niche” which promotes leukemic stem cell (LSC) function and impairs the maintenance of normal HSC. However, much remains to be understood about how different leukemic cells impacts the BM microenvironment and, in turn, how changes in the activity of specific BM niche cells contribute to AML pathogenesis. This talk will highlight some of the current understanding of the alterations of BM niche components and how the dialogue between leukemic and stromal cells participated in leukemogenesis.



INV08 Molecular regulation of muscle stem cell asymmetric division

M A Rudnicki 1 1: Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada Dystrophin is expressed in differentiated myofibers where it is required for sarcolemmal integrity, and loss-of-function mutations in its gene result in Duchenne Muscular Dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Recently it was reported that dystrophin acts as a tumor suppressor gene in malignant sarcomas associated with myogenic programs. We discovered that a subset of satellite cells in skeletal muscle are self-renewing stem cells that give rise to myogenic progenitors through asymmetric apical-basal cell divisions. Our identification of satellite stem cells has facilitated important insights into satellite cell biology. For example, we discovered Wnt7a/Fzd7 signaling as important intrinsic control mechanism that plays a central role in regulating the pool size of the satellite stem cell compartment by stimulating symmetric stem cell expansion. Direct injection of recombinant Wnt7a protein into muscle significantly augments regeneration. Wnt7a treated muscles were larger, contained higher numbers of satellite cells, larger caliber myofibers, and were able to generate more force upon stimulation. Thus, the regulation of asymmetric stem cell division is a key control point that impacts the efficacy of the entire regenerative program. Stem cell polarity is established by the PAR complex, comprised of PAR3/PAR6/aPKC, to regulate self-renewal and expansion. We have discovered that full-length dystrophin is expressed in satellite stem cells in skeletal muscle. We have made the seminal discovery that dystrophin regulates the establishment of PAR-mediated polarity in satellite cells. In the absence of dystrophin, the polarity effector Par1b is dysregulated, leading to the failure of Par3 to become localized to the cortex associated with the basal lamina. Importantly, this results in an abnormal increase in centrosome number, a 10-fold reduction in the numbers of satellite stem cells undergoing asymmetric divisions, and a marked decrease in the generation of myogenin-expressing progenitors. Accordingly, our data suggests that the failure of regenerative myogenesis to keep pace with disease progression in DMD is not due to muscle stem cell exhaustion, but rather is due to a cell-autonomous deficiency in asymmetric division. Deficiencies in asymmetric stem cell division have been associated with tumorigenesis, thus our experiments provide mechanistic insight into how dystrophin functions as a tumor suppressor gene in rhabdomyosarcoma.

INV09 Stem and progenitor cells in adult mouse mammary gland

M A Glukhova 1 1: UMR144 CNRS-Institut Curie, Research Center, 26 rue d’Ulm, 75248, Paris, France The mammary epithelium is organized in two cell layers, the luminal and the basal. Most basal cells express smooth muscle-specific contractile proteins and, therefore, they are termed as myoepithelial cells. During lactation, luminal cells produce and secrete milk, whereas basal myoepithelial cells contract to eject the milk from the body. Multipotent stem cells able to regenerate mammary gland upon transplantation reside in the basal layer of the mammary epithelium, whereas luminal compartment, devoid of the regenerative potential, harbors progenitor cells that give rise to ductal and secretory alveolar cells. The precise molecular characteristics of the mammary stem cells are not known, whereas the molecular pathways essential for mammary stem and progenitor cell function are poorly understood.

The aim of our work is to define and characterize specific microenvironment required for the maintenance of stem and progenitor cell populations in adult mammary epithelium, i.e., mammary stem cell niche. To this end, we study the role of the interactions between mammary epithelial cells and extracellular matrix (ECM), the mammary basement membrane. Integrins are ECM receptors present on the surface of mammary epithelial cells. We have found that integrins containing alpha 3 or alpha 6 subunits, binding cells to laminins, major constituents of the basement membrane, are essential for the function of mammary stem cells and mammary tumorigenesis.

INV10 Diversity of progenitors, lineage relationships and cell-cycle regulation during primate corticogenesis

Colette Dehay 1 1: Stem Cell and Brain Research Institute, Inserm U1208, Université de Lyon

Using 2 photon Time-lapse video recording coupled with immunolabelling on organotypic embryonic brain slices, we showed that deep and superficial neuron progenitors exhibit distinct lineages properties and cell-cycle kinetics in the



primate. Superficial neuron progenitors (generated by the outer Sub Ventricular zone-OSVZ) are characterized by a shortening of G1 and S phases compared with deep neuron progenitors (mostly generated by the Ventricular Zone-VZ), which largely departs from what has been observed in other species (Betizeau et al., Neuron 2013). RNA-seq analysis performed at the time of generation of superficial neurons, showed that newly evolved, primate-specific miRNAs uniquely characterize the distinct germinal zones (VZ, OSVZ) in the monkey. We found that a high proportion of these differentially expressed primate specific miRNAs target cell-cycle control genes and more specifically the G1/S transition regulators in supragranular neuron progenitors (Arcila et al. Neuron 2014). This evolutionary innovation in wiring of the gene networks that targets the G1 /S transition at the time of generation of superficial neurons is responsible for the dramatic expansion of the OSVZ, considered to underlie the complexity of the human and non human primate cerebral cortex (Dehay et al., Neuron, 2015).

This work is supported by the LabEx CORTEX (ANR-11-LABX-0042) and LabEx DEVweCAN (ANR-10-LABX-0061) of Université de Lyon (ANR-11-IDEX-0007), ANR PRIMACOR and the Fondation Recherche Médicale (équipe 2016)

INV11 Temporal identity progression in mouse retinal progenitor cells

M Cayouette 1 1: IRCM, Montréal, Canada The complexity of the central nervous system depends on the generation of large numbers of different neuronal subtypes. In the retina, this cellular diversity arises progressively from retinal progenitor cells undergoing temporal transitions in their competence to produce different cell types as developmental proceeds. In Drosophila neuroblasts, transcription factor cascades control this process, but how these ‘temporal identity factors’ act mechanistically remains largely unclear. We have shown previously that Casz1, the vertebrate ortholog of the Drosophila temporal factor castor, operates in multipotent retinal progenitor cells to promote the generation of cells born at mid/late stages of development like rod photoreceptors, while suppressing the generation of cells born during the early or latest stages of development, thereby defining a mid/late competence window. In this talk, I will present our most recent results indicating that Casz1 interacts with the polycomb repressor complex in a splice variant specific manner to control cell fate. Both Casz1 and polycomb knock-down in retinal progenitors have similar effects on cell fate. Moreover, while Casz1 overexpression leads to the increased production of rod photoreceptors and decreased production of Müller glia, these effects are lost when polycomb activity is simultaneously blocked. Finally, transcriptomics data indicate that Casz1 overexpression leads to the suppression of some polycomb targets and the upregulation of others, suggesting that Casz1 may switch polycomb targeting in a stage-specific fashion. Generally, these results support a model in which the competence of neural progenitors is controlled by dynamic changes in genome organization mediated by temporal identity factors.

INV12 Corneal epithelial cells derived from pluripotent stem cells as an alternative to animal eye toxicity test and ocular cell therapy

D Aberdam 1 1: Inserm 2: Paris Diderot 3: Inserm 4: Inserm Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetical products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Here we derivate limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged like adult somatic counterparts. Morever, comparative transcriptome and IF analysis demonstrate that they are morphologically and molecularly similar to the adult stem cells and similarly sensitive to cytotoxicity treatment. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetical and drugs tests and cell therapy.



INV13 Deciphering naive ground state pluripotency instruction in mouse embryonic stem cells

F Lavial 1 1: Cellular reprogramming and oncogenesis Laboratory - ATIP/Avenir Laboratory, Cancer research center of Lyon - INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, 69008 Lyon Understanding pluripotent stem cell biology is a fundamental goal in developmental biology, and research into diverse diseases. To achieve their great medical potential, a thorough understanding of the molecular features instructing pluripotency is required. The pluripotent state was first captured in mouse embryonic stem cells (mESC) over 30 years ago. The conventional mESC culture requires the presence of Leukemia Inhibitory Factor (LIF) and serum to maintain a self-renewing and pluripotent state. However, a pluripotent "ground state", bearing resemblance to preimplantation mouse epiblasts, can be established chemically through dual inhibition (2i) of the prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. However, the existence and the identity of endogenous molecules instructing naive ground state pluripotency remains unknown. Investigating whether and how distinct pluripotent states are endogenously triggered is a crucial objective to better understand preimplantation embryogenesis and somatic cell reprogramming. We will discuss recent findings about signaling pathways instructing naive ground state features acquisition in mESC.

INV14 Direct Reprogramming of Glial Cells into Functional Neurons: A New Avenue towards Brain Repair

C HEINRICH 1 1: Grenoble Institute of Neurosciences, INSERM U 1216 Direct cellular reprogramming or cell-fate conversion across cell lineages is an innovative approach toward cell-based therapies for regenerative medicine in various organs (brain, pancreas or heart). The conversion of non-neuronal cells into clinically relevant neurons emerges as a novel strategy to regenerate lost neurons for brain repair. We have explored a unique strategy aiming at reprogramming glial cells residing at the injury site into induced neurons (iNs), with the underlying rationale to recruit glia as an endogenous cellular source for brain repair. We showed in vitro that astroglia isolated from the mouse postnatal cortex can be reprogrammed into functional, synapse-forming neurons by forced expression of neurogenic transcription factors. Importantly, the neurotransmitter fate choice of iNs can be controlled by selective expression of distinct neurogenic factors: Neurogenin2 directs cortical astroglia to generate glutamatergic neurons, while Ascl1 and/or Dlx2 induces a GABAergic identity. Next, we showed that glia-to-neuron conversion can be translated to the context of the adult mouse brain in vivo and following injury. NG2 glia, proliferating in the cortex of adult mice in response to stab wound injury, can be instructed by Ascl1 and/or Sox2 to generate iNs that elicit action potentials and receive spontaneous synaptic inputs from endogenous neurons neighboring the injury site. This indicates that iNs are recognized by endogenous neurons as functional synaptic targets and are incorporated into local neuronal circuits. Importantly, we have also shown that human non-neuronal cells isolated from surgical specimens resected from the adult cortex can also be converted into functional iNs.

INV15 Therapeutic implications of cellular heterogeneity and plasticity in pancreatic cancer

C Heeschen 1 1: Barts Cancer Institute QMUL Pancreatic cancer is still a devastating diagnosis. Up to 90% of patients present with advanced disease and essentially all of them die within 12 months due to lack of effective maintenance treatments. The almost uniform occurrence of disease relapse can be attributed at least in part to the existence of pancreatic cancer stem cells and their distinct molecular features. Specifically, we have shown that

• pancreatic cancer is hierarchical organised and cancer stem cells as the root of this disease drive tumour progression, metastasis and chemoresistance. • pharmacological or genetic elimination of pancreatic cancer stem cells results in increased survival in preclinical mouse models. • cancer stem cells bear a distinct metabolic phenotype with limited plasticity that is amendable to therapeutic targeting. Circulating cancer stem cells are suitable for metabolic profiling and may be used for precision medicine approaches.



• the pancreatic tumour microenvironment acts as a cancer stem cell-promoting entity further enhancing stemness and chemoresistance of cancer stem cells.

Building on these findings and newly developed technology in our lab, we are now systematically determining, which common key regulators, intrinsic or extrinsic, control the process of stemness in pancreatic cancer. We are developing means to most efficiently target identified key regulators as part of a multimodal treatment strategy, which should aid in improving treatment response and preventing relapse.

INV16 Skin and Corneal Epithelial Stem Cells : Tissue-Engineered Substitutes for in vitro studies and patient treatment.

L Germain 1 1: Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX, Centre de Recherche du CHU de Québec, 1401, 18e rue, Québec, G1J 1Z4 and Département de chirurgie, Faculté de médecine, Université Laval Tissue engineering allows the reconstruction of 3D-tissue substitutes from cells and extracellular matrix with or without biomaterials. These substitutes offers new tools for in vitro studies to better understand complex process such as carcinogenesis. The presence of several cell types organize in a 3D environment favors the study of cell-cell and cell-extracellular matrix interactions. We have designed the self-assembly approach of tissue engineering to elaborate various 3D tissue substitutes that present good histological properties. Histological features vary depending on the cell type present in the construct. This is observed with normal cells as well as cancer cells. For example, metastatic cells cross the basement membrane to invade the stromal tissue whereas non-metastatic cancer cells do not. Moreover, stem cells are preserved in these tissues. In tissue-engineered skin (TES), a subset of epidermal basal cells exhibits the stem-cell associated slow-cycling property. An important proportion of these slow-cycling cells highly expressed keratin 19 (K19), and keratinocytes harvested from TESs gave rise to stem cell-like colonies in secondary monolayer subcultures. Moreover, we succeeded in producing substitutes comprising a capillary network. They revealed that interactions between epithelial cells and endothelial cells regulate capillary size. Finally, the tissue-engineered skin substitutes were used as autologous full-thickness grafts for the permanent coverage of full-thickness burn wounds on patients. The integrity of the transplanted TES persisted over time confirming the presence of functional stem cells. These results indicate that the microenvironment supports epithelial stem cell survival and function within TES in vitro and after grafting in patient in vivo.

INV17 Plasticity and chemoresistance in T-ALL: a question of microenvironment

F Pfulmio 1 1: Laboratoire des cellules souches hématopoïétiques et des leucémies, UMR 967 INSERM/CEA Fontenay-aux-Roses, France T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell progenitor malignancy, which mainly affects children and young adults. In T-ALL development, leukemic cells home to various bone marrow (BM) sites that include adipocyte-poor (red marrow) and adipocyte-rich (yellow marrow) niches. Using xenografts of human and mouse T-ALL, we explored the impact of these distinct BM sites on leukemic cells. We demonstrate that T-ALL cells invade all studied BM sites but with different kinetics depending on those sites. T-ALL cells also displayed BM-niche specific characteristics in terms of cell surface phenotype, metabolism, cell cycle progression albeit genomic abnormalities were similar wherever the T-ALL cells were isolated from. Overall, our current results demonstrate that distinct BM sites differentially orchestrate T-ALL development and during my talk I will also show how such BM niches may participate into chemo-resistance.

OR01 Unveiling the mechanisms of the RNA-binding protein Musashi1 in stemness and drug resistance of intestinal epithelial cells

C Frau 1 M Godart 1 C Le Nevé 1 S Ansieau 1 M Plateroti 1 1: Cancer Research Center of Lyon

Background. The RNA-binding protein Musashi1 (Msi1) plays a role in stem cell biology of several tissues including the intestinal epithelium. In addition a link between Msi1 expression levels and intestinal tumor stage or with drug



resistance has also been observed. With the aim to investigate the mechanisms of Msi1 function on intestinal stem cells and drug resistance, we took advantage of the v-Msi1 mice recently developed in our laboratory, harboring an ectopic Msi1 expression in the whole intestinal epithelium. Results. We used an approach of 3D-organoid cultures from WT and v-Msi1 crypts treated or not with 1.5 ug/mL 5-FU, a drug commonly used to treat intestinal cancers. We first assessed the number of viable organoids and their growth characteristics at different time-points after treatment. Interestingly, 5-FU treatment strongly reduced the growth and viability of WT but was inefficient on v-Msi1 organoids. In order to link the effects on organoid's growth with drug resistance and stemness, we analysed the expression of enzymes involved in 5-FU metabolism and action and the role on Lgr5-positive stem cells. Contrariwise to the WT condition, the v-Msi1 organoids presented a drug resistant phenotype, as indicated by the expression pattern of 5-FU metabolizing enzymes, and that stem cells were not affected by 5-FU treatment. In conclusions. These results suggest that Msi1 is a key regulator of both stemness and resistance to 5-FU. Our next objectives will be an in depth investigation of this process in animals in vivo in both physiological conditions and in tumors.

OR02 Rethinking regeneration

A Izeta 1 1: Biodonostia Institute Tissue regeneration proceeds in different ways. Most researchers focus in adult "tissue resident" stem cells differentiating into committed precursors to replace the damaged area. However the organism has alternatives such as plasticity of "stably differentiated" cells, in the form of dedifferentiation to a progenitor state, or transdifferentiation/reprogramming between committed cell types. The physiological role of these alternative strategies is beginning to emerge in a number of tissues. Thus the Waddington landscape model may be replaced by new concepts such as metastable cellular states, as proposed by Sieweke. In the lab we are interested in understanding cellular plasticity vs stemness in dermal regeneration and repair; and in particular we have contributed to clarify the role of dedifferentiated Schwann cells (SC) and pericytes in tissue homeostasis and wound healing. We think that SC not only may transition to other cell types (as required by local circumstances) but also act as paracrine signaling hubs to promote regeneration or repair, or act as a "direction guide" similar to their role in peripheral nerve regeneration. These phenomena may also happen in repair of other tissues. Similar mechanisms may underlie the cancer associated stromal remodeling that mediates invasion of several tumor types.

OR03 A glucocorticoid-dependent metabolic program supports cancer stem cell properties in breast cancer

E Nouri 1 6 7 8 9 L Payen-Gay 1 5 6 J Guitton 5 6 E Vulliet 2 P Jame 2 A Bulete 2 M Devouassoux-Shisheboran 5 C Machon 5 6 M Faivre 4 R Ferrigno 4 E Charafe-Jauffret 3 C Ginestier 3 A Puisieux 1 6 7 8 A M Vigneron 1 6 7 8 1: Cancer Research Center of Lyon 2: Institut des Sciences Analytiques 3: Cancer Research Center of Marseille 4: Institut des Nanotechnologies de Lyon 5: Hospices civils de Lyon 6: Université Claude Bernard Lyon1 7: Inserm U1052 8: CNRS U5286 9: Clevexel Cancer cells with stemness properties, generally designated as cancer stem cells (CSCs) are at the apex of the hierarchical organisation of tumours. CSCs are considered to drive tumour recurrence and metastasis formation. However, how they conciliate in these contexts their neoplastic activities with a nutritive environment different from the one found in an established tumour is unknown. Here we unveil the prominent role of glucocorticoid activity in the control of mammary cancer cell plasticity and the induction of metabolic pliancy. By regulating a MAFB-dependent cell reprogramming, glucocorticoids control stemness traits in malignant epithelial cells. As an integral part of this reprogramming, glucocorticoids activate the hexosamine biosynthetic pathway, modify cancer cell “metabotype”, and increase anabolic efficiency. Glucocorticoids are thus master regulators of cancer cell fate specification and tumorigenic potential. These data suggest that the inhibition of their metabolic activity could constitute an original strategy for CSC eradication and tumour treatment.



OR04 BMP pathway's involvement in leukemic stem cells resistance

E GROCKOWIAK 1 2 3 B LAPERROUSAZ 1 2 3 S JEANPIERRE 1 3 4 T VOELTZEL 1 2 3 S GOBERT 1 2 3 F E NICOLINI 1 3 5 V MAGUER-SATTA 1 2 3 1: Inserm U1052, CNRS UMR5286, Cancer Research Center of Lyon 2: University of Lyon 3: Team : BMP, tumor niche & resistance 4: Centre Léon Bérard 5: Centre Hospitalier Lyon Sud, Hematology Department Chronic myelogenous leukemia (CML) arises from a genetic translocation in a hematopoietic stem cell (HSC), which induces the expression of an oncogenetic tyrosine kinase BCR-ABL. Tyrosine Kinase Inhibitors (TKI) that inhibit specifically BCR-ABL have revolutionized medical care of patients. However, they are unable, in most cases to prevent relapse following treatment arrest, likely due to the persistance of leukemic stem cells (LSC) that are resistant to TKI, by mechanisms that remain poorly understood. The Bone Morphogenetic Protein (BMP) pathway regulates HSC properties. Deregulations of this pathway is a crucial driver of the early steps of CML development; indeed, before TKI treatment, BMPR1b is overexpressed in leukemic cells, and the tumor niche secrets higher level of BMP2 and BMP4 cytokines. These BMP induce LSC and leukemic progenitors maintenance and expansion. Here, we explore the persistence of BMP pathways alterations in more than 140 CML patients, and their importance towards treatments efficiency. Our results demonstrate that, conversely to patients in remission, resistant cells of patients under treatment present sustained high levels of BMPR1b in immature cells combined with high rates of soluble BMP. BMP2/4 induce the resistance of LSC with high BMPR1b expression. This mechanism seems mediated by the BMP induction of TWIST-1 expression, previously identified by our team as a predictive factor of TKI resistance. An autocrine loop of BMP2/4 could induce the resistance in leukemic immature cells of resistant patients. Altogether, our data clearly establish that BMP pathway alterations trigger CML primitive cells amplification and are involved in their resistance.

OR05 The role of oriented cell division in prostate progenitor cell homeostasis

M ER Shafer 1 3 M Tremblay 1 3 M Lacomme 2 C Monat 2 4 M Cayouette 2 4 M Bouchard 1 3 1: Goodman Cancer Centre 2: Institute de Researches Cliniques de Montreal 3: Department of Biochemistry, McGill University 4: Universitie de Montreal The formation of multiple lineages and the stratification of cell types during epithelial morphogenesis is controlled by extrinsic and intrinsic cell fate determination signals. The epithelial lineages and stratified architecture of the prostate are generated during development by coordinated cell divisions in bi-potent basal stem cells. These divisions control the relative amounts of the stem-like basal, and differentiated luminal cells. Using conditional gene inactivation in the mouse, we have observed that the transcription factor Gata3 controls this process by regulating the localization of the protein kinase aPKC independently of the Par complex. Deregulation of aPKC by loss of Gata3 leads to randomization of spindle orientation in basal stem cells, and an increase in the formation of ‘double-positive’ progenitor cells. These defects ultimately lead to epithelial hyperplasia, without affecting the intrinsic proliferation rate of the epithelium or the individual cell types. We further show that disrupting the interaction between aPKC-Par6 is sufficient to recapitulate the spindle and cell lineage phenotypes. aPKC controls spindle orientation by directly interacting with the spindle complex protein LGN, which links the spindle with cortical membrane during cell divisions. In contrast to its necessity for asymmetric cell divisions in the epidermis, we recently found that LGN is required for symmetric divisions in prostate stem cells. Loss of LGN leads to epithelial hyperplasia and increases in progenitor cells. Collectively, these results identify a critical role for Gata3 in lineage specification in the prostate, and further highlight the importance of regulating spindle orientation for hierarchical cell lineage organization.

OR06 Deficit in myeloid lineage and very early defect in HSPC pool: an embryonic origin of Fanconi haematological disorders

C Domenech 1 2 3 A Rousseau 4 L Maillard 1 2 L Petit 1 D Clay 5 S SanFilippo 1 J Soulier 4 M Souyri 1 2 1: laboratoire de Biologie du Développement, CNRS UMR 7622, Inserm U1156, IBPS,UPMC 2: Inserm UMR-S1131, IUH Paris 7 3: IHOP, UCBL, HCL, Lyon 4: Inserm U944, IUH, Hopîtal St Louis, Paris7 5: IAL/ INserm U1197, Villejuif



Fanconi anemia (FA), a genetic disorder due to mutations in FANC genes involved in DNA repair, leads to bone marrow failure (BMF) early during childhood, and strongly predisposes to acute myeloid leukaemia (AML). To date, mechanisms of this disease development remain misunderstood. Bone marrow (BM) hypocellularity that often precedes clinical symptoms, coupled with the presence of developmental abnormalities at birth, led us to speculate that FA haematological disorders might have an embryonic origin. In order to answer these questions, we carried out studies on Fancg-/- mouse embryos and human FA fetuses. Our results uncovered a quantitative HP defect early during Fancg-/- embryonic development, both in fetal liver (FL) and placenta (PL), as well as an important HSC defect, as demonstrated by in vivo long-term hematopoietic reconstitution (LTR) assays. This HSC defect is accompanied by an overall defect of terminal hematopoietic lineages, especially myeloid. Transcriptomic analysis of Fancg-/- FL and Pl HSCs is under investigation. Interestingly, we also observed an HP/HSC defect in a human FA FL (13.5 weeks of gestation), associated with a total lack of in vitro amplification compared to control FL. Taken together, these data demonstrate that a profound and significant deficit of HSC and progenitors cells is present since the earliest stages of embryonic development in FA. Altogether, our work suggests a major role of the Fanconi pathway in the development of hematopoietic system with a deficit of amplification of HSC.

OR07 Involvement of YB-in oncogenic KRAS-induced de novo human breast cancers

S Lefort 1 A M El-Naggar 1 D Pellacani 1 L Nguyen 1 P H Sorensen 1 C J Eaves 1 1: The University of British Columbia Spontaneously arising human breast cancers are expanding clonal outgrowths of cells that are originally present in the normal adult human mammary gland and have acquired abnormal properties of invasiveness and blockade of normal differentiation. Accumulating data indicate that by the time most breast cancers are diagnosed, they have already diversified both genetically and biologically in terms of their subclonal properties and representation in any given sample. Indeed, their complete characterization is generally impossible which is a likely explanation for historic and continuing difficulties to devise effective therapies. This situation has prompted interest in the creation of experimental systems in which the first events that establish a tumorigenic state in primary human cell types and then define their progression are accessible to analysis. We recently showed that such an in vivo model can be generated at high efficiency by transducing purified basal or luminal progenitors isolated from normal human mammary tissue with a single oncogene (KRASG12D) and then immediately transplanting the cells into immunodeficient mice (Nguyen et al, Nature 2015). Histological and molecular characterization of these developing tumours has now shown that elevated and ubiquitous expression of cold shock domain protein YB-1 becomes apparent within 2 weeks, regardless of the cell type transduced and is stable upon subsequent tumour passaging. We have also found that in this model, YB-1 inhibition impairs tumour initiation and at later times inhibits metastatic ability. These experiments demonstrate the power of de novo models of human breast cancer to enable critical early events to be identified.

OR08 Age-associated alterations in haematopoietic stem cell heterochromatin

M GOODHARDT 1 2 D Djeghloul 1 2 F Porteu 4 I Naguibneva 5 D Garrick 1 2 1: INSERM UMRS1126 2: Université Paris Diderot 3: INSERM U1009 4: INSERM UMRS1170 5: INSERM UMRS967 All myeloid and lymphoid blood cell lineages are continually replenished throughout adult life from a reservoir of rare multipotent hematopoietic stem cells (HSC) residing in the bone marrow. The capacity of HSC to generate antibody-producing B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.



OR09 PUMILIO/FOXP1 signaling drives maintenance of hematopoietic stem/progenitor and leukemia cells

C Naudin 1 2 3 4 A Hattabi 1 2 3 4 F Michelet 1 2 3 4 A Miri-Nezhad 1 2 3 4 A Ben Youcef 5 6 F Pflumio 5 6 F Guillonneau 1 2 3 4 S Fichelson 1 2 3 4 I Vigon 1 2 3 4 I Dusanter-Fourt 1 2 3 4 E Lauret 1 23 4 1: Inserm U1016 2: Institut Cochin 3: CNRS UMR8104 4: Université Paris Descartes 5: UMR967 6: CEA RNA-binding proteins (RBPs) have emerged as important regulators of Invertebrate adult stem cells, but their activities remain poorly appreciated in mammals. We demonstrated that the two mammalian RBPs PUM1 and PUM2, members of the PUF family of post-transcriptional regulators, are essential for hematopoietic stem/progenitor cell (HSPC) maintenance in vitro and in vivo upon reconstitution assays. PUM1/2 regulate cell cycle and survival of HSPCs. Interestingly, PUM1/2 expression is enhanced in the most immature samples of acute myeloid leukemia (AML), and promote leukemic cell growth. To identify PUM targets, we performed a comparative and quantitative global proteomic analysis of progenitor cells that expressed normal or low levels of PUM1 or PUM2. We identified the FOXP1 transcription factor as a new target up-regulated by PUM1/2. Given that nothing is currently known on FOXP1 in early hematopoiesis, we have analyzed its function in HSPCs. We showed that FOXP1 supports HSPC maintenance and leukemic cell growth, and represses expression of p21CIP1 and p27KIP1 cycle inhibitors, which mimics PUM1/2 activities. The expression of FOXP1 is strongly correlated with PUM1 and PUM2 levels in HSPCs and AML cells. Moreover, FOXP1 expression partially rescued shPUM antiproliferative and pro-apoptotic activities. Finally, we observed that PUM1/2 enhance FOXP1 expression through direct binding to two canonical PUM responsive elements in FOXP1-3’UTR. Our results reveal a novel regulatory pathway, underscoring a previously unknown and interconnected major role of PUM1/2 and FOXP1 in the maintenance of normal and leukemic HSPCs, and provide with new putative predictive markers and potential therapeutic targets of hemopathies.

OR10 Pre-cancerous mammary stem cells involved in metastatic colonization of the lung.

F Kabeer 2 C Darini 2 P O Bachant-Winner 2 B Haibe-Kains 2 P Chavrier 1 A Vincent-Salomon 1 G Darrasse-Jeze 3 K Podsypanina 1 2 1: Institut Curie 2: Institut de recherches cliniques de Montereal 3: Hôpital Necker-Enfants Malades Normal and tumor human breast cells from patients with ductal carcinoma in situ (DCIS) can grow in immune-compromised mice when injected inside mouse mammary milk ducts. Intriguingly, despite their ostensibly harmless status, cells from some DCIS patients can also establish experimental metastases in the lung of immunodeficient mice. To better understand the ectopic tissue colonization by pre-malignant cells, we isolated and characterized a lung-colonizing cell (LCC) subset of the mammary gland. The LCCs formed up to ten times more outgrowths after intravenous injection than the unsorted mammary cells, and displayed phenotypic and functional characteristics of stem cells, including the pattern of surface marker expression, asymmetric division, self-renewal and generation of multilineage progeny. Despite their non-cancerous status, the LCCs expressed a gene signature that predicted poor metastasis-free survival in women with breast cancer, drawing a link between regenerative and malignant properties in the breast epithelium. Indeed, when mouse mammary cells were induced to overexpress a potent human oncogene, ERBB2 (Her2/Neu), the injected LCCs produced mammary tumors directly in the lungs, but none of the other mammary subsets did. After repeated cycles of ERBB2 induction and deinduction, the LCCs have survived and produced recurrent tumors, suggesting that LCC-like properties may contribute to the development of metastatic disease and cancer recurrence. Finally, in the absence of transgenic oncogene induction, the ectopic mammary cells displayed markers of DNA-damage response, suggesting a possible mechanism for spontaneous transformation in LCC.

P01 High expression of embryonic transcription factor OCT¾ associated with differentiation anomalies in acute myeloid leukemia and myelodysplastic syndromes

T PICOT 1 A FAYARD 1 C AANEI 1 S KESR 1 Y WU 1 E TAVERNIER-TARDY 1 P FLANDRIN-GRESTA 1 S TONDEUR 1 M GOUTTENOIRE 1 E WATTEL 1 D GUYOTAT 1 L CAMPOS-CATAFAL 1 1: Laboratoire d'Hématologie



Acute Myeloid Leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by the expansion and resistance to apoptosis of poorly differentiated myeloid cells. Long term propagation of the disease is mainly due to a subset of cells termed Leukemic Stem Cells (LSCs), which reacquire self renewing properties and give rise to the leukemic clone. Several studies have suggested that LSC mostly belong to the CD34+CD38- compartment. Self-renewal and lack of differentiation are also features of embryonic stem cells, characterized by the expression of specific genes like the transcription factor OCT¾. We investigated the potential role OCT¾ in leukemogenesis by evaluating its expression in stem cells from AML and MDS patients compared to normal HSC subsets. We studied 139 AML and 42 MDS newly diagnosed cases by Multicolor Flow Cytometry (MFC) and RT-PCR. We also evaluated in HL60 leukemic cell line its potential involvement in myeloid differentiation by inhibiting its expression by shRNA. In MCF, we observed an up-regulation of OCT¾ in AML cells as compared to normal cells. This expression was higher in CD34+CD38- than in CD34+CD38+ subsets in normal as well as in leukemic marrows. Furthermore, an over-expression of OCT¾ was observed in MDS, more specifically in forms with excess of blasts. Finally, HL60 differentiation with retinoic acid induced a decrease of OCT¾ expression. Inhibition experiments by shRNA showed a strong downregulation of OCT¾ and an arrest of proliferation associated with myeloid differentiation. These results suggest that aberrant expression of OCT¾ in AML and MDS is associated with differentiation blockade.

P02 miR-29a inhibition reduce esterogen receptor alpha in breast cancer

G R Khamisipour 1 M Rahnama 1 S Akbarzadeh 1 B Naeimi 1 M Hasanpour 1 Z Mansourabadi 1 A Moazzeni 1 S Shamsyian 1 1: Bushehr University of Medical Sciences,Bushehr,Iran Introduction: Breast cancer is the most leading cause of cancer among women worldwide. MicroRNAs(miRNAs) are small non-coding RNA which plays critical role in variety of cellular function such as cell proliferation and apoptosis. Recent Studies demonstrated, miRNA-29a overexpressed in patients serum, which might be correlated with pathogenesis of breast cancer. It has been reported that estrogen and its receptor (ERα) leads to progression and cellular proliferation. In current study, we assessed the inhibitory effect of anti-miRNA-29a on expression of Estrogen receptor-α in MCF-7 breast cancer cell line. Material and Methods: MCF-7 cells were cultured in High Glucose DMEM supplemented with 10 % Fetal Bovine serum (FBS). This study was performed in four different groups including control, anti-miR-29a treated, Anti-miR-29a combined with Taxol and Taxol alone. After treatment total RNA was extracted and cDNA was synthesized. Finally, expression level of ERα was assessed using quantitative Real time PCR (qRT-PCR) in different groups. Results: We observed significant differences in level of ERα between all three groups, anti-miR-29a 27%, taxol 42% and antimir29a + taxol 54% level of ERα (p<0.05). Conclusion: Our results showed that anti-miR29a could be used as a non invasive targeted therapeutic agent in treatment of breast cancer in combination with other chemotherapy regiments such as taxol. Key words: Breast cancer, miR-29a, Estrogen receptor, Apoptosis

P03 Studying stem cells population dynamics and tracing cellular fate with iPLEX mass spectrometry.

A Bernat 1 2 K Meller 1 I Aksoy 3 P Savatier 3 K P Bielawski 1 1: Laboratory of Molecular Diagnostics, Laboratory of Genetic Analysis, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk & Medical University of Gdansk, Gdansk, POLAND; 2: Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, POLAND 3: INSERM U1208, Stem Cell and Brain Research Institute, Laboratory of Pluripotent Stem Cells, Bron, FRANCE The great promise that embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) hold for cell replacement therapies is hampered by their high heterogeneity and genomic instability. Genetic labelling technologies shed some light on the dynamics of stem and progenitor cell fate determination during development, organogenesis and tumorigenesis, however applied methods used to identify the genetic labels are time-consuming, labour-intensive and require challenging analytical and computational steps for their identification. Here, we report the novel strategy based on mass spectrometry (MS), with greatly reduced complexity and time of analysis, for detection of DNA labels (barcodes) in stem cell population. iPLEX MALDI-TOF MS, conventionally used for high-throughput diagnostic genotyping, has been applied to detect designed DNA barcodes, introduced into mouse embryonic stem cells by means of viral infections. All introduced DNA barcodes are easily detected by iPLEX MALDI-TOF MS during routine cell culture. By mixing barcoded cell line showing growth advantage with normal barcoded population, we have studied the dynamics of cell culture repopulation. We were able also to detect single genetic labels in late blastocyst



after introduction of barcoded cells into 8-cell stage mouse embryo. Apart from short time-to-result and simple analysis, it is a versatile tool for studying population dynamics and controlled capture of single cell progeny. The developed method can be applied for indirect monitoring of genetic stability in the population of iPSCs, as decrease in genetic diversity may reflect in gradual enrichment of mutant cells and concomitant accumulation of chromosomal aberrations.

P04 Development of fast detection method for most common aneuploidies in a population of induced pluripotent stem cells (iPSC), with iPLEX® MALDI-TOF MS.

K Meller 1 A Bernat 1 2 K P Bielawski 1 1: Laboratory of Molecular Diagnostics, Laboratory of Genetic Analysis, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, POLAND 2: Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec /n. Warsaw, POLAND Existing restrictions on the use of induced pluripotent stem cells (iPSC) in regenerative medicine, result from the emerging signs of their genetic instability, due to the adaptation and long propagation to in vitro cell cultures. The objective of this project is to develop fast and reliable method for detection of the most common aneuploidy in the population of iPSC cells - trisomy of chromosome 12, with iPLEX® SAR MALDI-TOF MS technique. This high-throughput method, based on genotyping with iPLEX® technology and relative quantification of alleles ratio deviation for each sample in the population for particular locus, allows for detection of putative CNVs localised within selected polymorphic sites. Using Hap Map database, we determined 359 heterozygous (MAF = 0.5), polymorphic sites located on chromosome 12, for Caucasians, and first 20 SNPs were selected. DNA from cells with defined trisomy of chromosome 12 and DNA with normal karyotype were used for needs of test development. The analysis of mass spectra from trisomy 12 chromosome DNA samples in comparison to the mass spectra from normal DNA, reveals a difference in intensity of the mass peaks obtained with the selected SNPs. First experiments allowed us to identify several informative polymorphic sites and will help further design the diagnostic test. The project has high scientific value as there is a need for fast and reliable diagnostic test for common abnormalities in iPSCs.

P05 Baboon envelope pseudotyped lentiviral vectors: a highly efficient new tool to genetically manipulate T-cell acute lymphoblastic leukaemia-initiating cells.

C Costa 3 G Hypolite 2 O Bernadin 1 C Lévy 1 F L Cosset 1 V Asnafi 2 E Macintyre 2 E Verhoeyen 1 M Tesio 2 1: CIRI, INSERM U1111, EVIR 2: INEM, INSERM) U1151, Laboratory of Onco-Hematology 3: CIRI, INSERM U1111 The malignant transformation of normal T-cell progenitor cells into self-renewing leukaemia-initiating cells causes the development of T-cell Acute Lymphoblastic Leukaemia (T-ALL), an aggressive hematologic cancer presenting unfavourable clinical features. Leukaemia-initiating cells are linked to chemotherapy resistance and relapse but the lack of tools to manipulate them prevents identification of their immunophenotypic and molecular features, thus precluding the development of novel targeted therapies. Our study reveals that lentiviral vectors (LVs) pseudotyped with a baboon retrovirus envelope (BaEV-LVs) are excellent tools to genetically manipulate T-ALL leukaemia initiating cells. These LVs enabled high-level transduction at low multiplicity of infection (MOI=10) since they use ASCT1 and ASCT2, two aminoacid transporters which are highly expressed on the T-ALL cells. The transduced blasts engrafted cohorts of primary and secondary NOD/SCID gamma mice, with typical T-ALL features, thus demonstrating that BaEV-LVs target leukaemia-initiating cells. Competitive xenografts models demonstrated that the BaEV-LV do not alter leukemic self-renewal, as the transduced blasts did not acquire a growth advantage, nor were they outcompeted by untransduced cells. Furthermore, our BaEV-pseudotyped lentiviral vectors will facilitate chimeric antigen receptor expression and the development of genomic editing strategies for primary T-ALL cells. BaEV-LVs are thus excellent tools to genetically manipulate leukaemia-initiating cells to aid revealing novel therapeutic avenues.



P06 Tumor cells with neuronal intermediate progenitor features define a subgroup of 1P/19Q co-deleted anaplastic gliomas

F Bielle 2 F Ducray 3 K Mokhtari 5 C Dehais 6 H Adle-Biassette 4 C Carpentier 5 A Chanut 6 M Polivka 4 S Poggioli 5 S Rosenberg 5 M Giry 5 Y Marie 5 C Duyckaerts 2 M Sanson 2 _ POLA Network 6 D Figarella-Branger 1 A Idbaih 2 1: Aix-Marseille University 2: UPMC - Paris 6 3: Université Claude Bernard Lyon 1, Lyon, France 4: AP-HP, Hôpital Lariboisière 5: Inserm U 1127, CNRS UMR 7225, Sorbonne Universités, UPMC Univ Paris 06 UMR S 1127, Institut du Cerveau et de la Moelle épinière, ICM, F-75013, Paris, France 6: AP-HP, Hôpitaux universitaires Pitié Salpêtrière Charles Foix Introduction Anaplastic oligodendroglial tumors (AOT) exhibit intertumor heterogeneity. Molecular biology of AOT has been deciphered over the last decades identifying three histomolecular subgroups with clinical relevance based on 1p/19q co-deletion and IDH statuses. In contrast, cell portrait of AOT is less well-known. Materials and Methods The present study is focused on intertumor and intratumor, cell lineage-oriented, heterogeneity in 1p/19q co-deleted AOT and is based, on pathological, transcriptomic and immunophenotypic studies in a training cohort and a validation cohort. Results A novel subgroup of newly diagnosed 1p/19q AOT overexpressing neuronal intermediate progenitor (NIP) genes was identified. This NIP overexpression pattern in AOT is associated with: (i) morphological and immunohistochemical similarities with embryonic subventricular zone, (ii) proliferating tumor cell subpopulation with NIP features including expression of INSM1 and no expression of SOX9, (iii) mutations in critical genes involved in NIP biology and, (iv) increased tumor necrosis. Interestingly, NIP tumor cell subpopulation increases in AOT recurrence compared to paired newly-diagnosed tumor. Conclusion Our results emphasize intertumor and intratumor heterogeneity in AOT and identified a tumor cell subpopulation exhibiting NIP characteristics that is potentially critical in oncogenesis of AOT. A better understanding of spatial and temporal intratumor cell heterogeneity in AOT will open new therapeutic avenues overcoming resistance to current anti-tumor treatments.

P07 Controlled vectorization of core pluripotency transcription factors in human cells by a new cell-penetrating peptide

B CAULIER 1 2 L BERTHOIN 2 F GARBAN 1 2 M C DAGHER 2 J L LENORMAND 2 B POLACK 2 B TOUSSAINT 2 D LAURIN 1 2 1: EFS Rhône-Alpes-Auvergne 2: TheREx TIMC UMR 5525 CNRS Somatic cell fate reprogramming by ectopic Transcription Factors (TFs) expression holds a great promise in disease modelling and regenerative medicine. To date, technologies used to drive dedifferentiation or transdifferentiation rely almost exclusively on the use of genetic material transfer. Although viruses or engineered plasmids are very efficient for this purpose, they raise important safety concerns, challenging the clinical transfer of cell fate technologies. We have developed efficient recombinant technologies to deliver therapeutics into eukaryote cells. One of those is based on the fusion of a cell-penetrating peptide (CPP) from the Epstein-Barr virus ZEBRA protein (named MD11) to the protein of interest. This CPP is able to translocate high molecular weight proteins in an endocytosis-independent mechanism. We fused: Oct4, Sox2, Klf4, c-Myc, Nanog TFs and the microRNA biogenesis regulator Lin28 as proteins of interest to MD11. We have successfully purified the independent MD11-Oct4, MD11-Sox2, MD11-Nanog and MD11-Lin28 proteins (named MD11-OSNL). The MD11 domain does not interfere with Oct4 association to its DNA consensus sequence. Moreover, MD11-Oct4 can be localized to the nucleus of in vitro treated cells in three hours. In a context of reprogramming experiments, the combination of repeated treatment with MD11-OSNL leads to the transcriptional activation of target genes. Overall, these results paved the way to a safe and clinically compliant vector. We are now working at inducing pluripotency and hematopoietic transdifferentiation from differentiated cells.

P08 Peptides derived from the dependence receptor ALK are proapoptotic for ALK-positive tumors

A Aubry 1 3 S Galiacy 1 4 F Lopez 1 R Bremner 3 C Racaud-Sultan 1 F Malecaze 1 2 4 M Allouche 1 2 1: Inserm 2: University of Toulouse 3: University of Toronto 4: Hopital Purpan Toulouse



ALK is a receptor tyrosine kinase with an oncogenic role due to constitutive activation of the kinase in various types of human malignancies. Unfortunately targeted therapy using ALK kinase inhibitors invariably leads to resistance. We previously demonstrated ALK is a dependence receptor. This implied that in contrast to kinase-activated ALK which stimulates proliferation, kinase-inactive ALK triggers or enhances apoptosis. Searching for new tools to target ALK, we synthesized peptides mimicking the proapoptotic domain of ALK and investigated their biological effects on tumor cells. We found that an ALK-derived peptide of 36 amino acids (P36) was cytotoxic for ALK-positive anaplastic large cell lymphoma and neuroblastoma cell lines. In contrast, ALK-negative tumor cells and normal peripheral blood mononuclear cells were insensitive to P36. The cytotoxic effect was due to caspase-dependent apoptosis and required N-myristoylation of the peptide. Two P36-derived shorter peptides as well as a cyclic peptide also induced apoptosis. Surface plasmon resonance and mass spectrometry analysis of P36-interacting proteins from two responsive cell lines, Cost lymphoma and SH-SY5Y neuroblastoma, uncovered partners that could involve p53-dependent signaling and pre-mRNA splicing. Furthermore siRNA-mediated knockdown of p53 rescued these cells from P36-induced apoptosis. Finally, we observed that a treatment combining P36 with the ALK-specific inhibitor crizotinib resulted in additive cytotoxicity. Therefore ALK-derived peptides could represent a novel targeted therapy for ALK-positive tumors. References Aubry A et al. Cell Death Disease 2015 Allouche M. Atlas Genet Cytogenet Oncol Haematol 2010 Allouche M. Cell Cycle 2007 Mourali J et al. Mol Cell Biol 2006

P09 Proliferation of cancer stem cells in ALDH-positive triple-negative breast cancer

S V Sazonov 1 2 A A Brilliant 1 Y M Brilliant 1 1: Institute of Medical Cell Technologies, Ekaterinburg, Russia 2: Ural State Medical University, Ekaterinburg, Russia The aim of the study was to estimate the proliferation level of cells in triple-negative breast cancer (TNBC) with high content of cancer stem cells. Material and methods: 55 cases of TNBC were studied. Immunohistochemical method was applied. Presence of ALDH1 expression was explored for detection of stem cells in cancer cells population. Rabbit Monoclonal Anti-Human ALDH1A1 (EP168) (Epitomics, USA) antibodies were used. Evaluation of ALDH1 expression was conducted by detection of number of stained cancer cells: 3+ (stained cells number is ≥50%), 2+ (stained cells number is < 50%, but >10%), 1+ ((stained cells number is ≤10%). Level of proliferation was evaluated by detection of nuclear index of cancer proliferation – Ki-67. Rabbit Monoclonal Anti-KI-67 (SP6) (Spring, USA) antibodies were used. Immunohistochemical tests were conducted by using of “Ventana” autostainer, USA. For detection of Ki-67 expression in cancer stem cells the double system of detection ultraView Universal AP Red/Universal DAB Detection Kit (Ventana, USA) was used. Results: 5 cases with high level (3+) of ALDH1 expression were found. It was found that cell population of TNBC in 9% contains more than 50% of cancer stem cells. Cancer stem cells of TNBC had an average index of proliferation – 71%, the average index of proliferation of cells which do not express ALDH1 was 58%. The differences between the groups were significant (p<0.05). Accordingly, it was found that cancer stem cells proliferative activity of ALDH1 (3+) cases of triple-negative breast cancer was higher than proliferative activity of cancer cells main pool.

P10 Deciphering the CD10 code in breast cancer reveals a molecular signature to identify cells prone to transformation and that sustain tumor heterogeneity

F Clément 1 X Schmidt 1 J P Foy 1 P Saintiny 1 A Beaumont 1 E Delay 1 S Jeanpierre 1 W Q Gao 2 H Zhu 2 B Guyot 1 V Maguer-Satta 1 1: CNRS UMR5286 INSERM U1052, UCBL, Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon 2: Renji hospital clinical stem cell research center, Shangai, China. We developped a model of early human breast epithelial progenitors cells transformation by exposure of the MCF10A cell line to BMP2/IL6. We observed that transformation is associated with an increase in CD10 expression. CD10 is a trans-membrane protease able to cleave peptides from the microenvironment and that interact with intracellular signalling pathway through PTEN. Previous results indicate that CD10 controls the fate of SC and is deregulated in breast cancer. We also showed that CD10 membrane expression allows the maintenance of immature cells, partly through its enzymatic function that inhibits mammary stem cells differentiation. As CD10 has been implicated in breast cancer initiation, progression and resistance, we tested the role of CD10 in our model of breast epithelial cells early transformation. In this model, the heterogenity of CD10 expression observed in breast tumors is recapitulated. Only CD10+ cells are able to regenerate both CD10+ and CD10- subpopulations and thus sustain initial tumor heterogeneity. We showed that the transformation mediated by BMP2/IL6 mainly target CD10+ cells. Modulating



CD10 expression has no impact on the transformation of our models, indicating that CD10 is more a marker of a population prone to transformation rather than a driver. We then performed a transcriptomic analysis on CD10+ and CD10- subpopulations. We identified a CD10 associated gene signature in normal cells of our model, which is overexpressed in their transformed counterparts but no longer CD10 associated. Expression of this CD10 signature increase with breast cancer aggressivness and is associated with lower patient survival.

P11 High PKNOX2 level of mesenchymal stem cells prompts hematopoietic stem cell differentiation towards myeloid lineages in coculture experiments

I Cagnan 1 F Aerts Kaya 1 D Uckan-Cetinkaya 1 A Gunel-Ozcan 1 1: Hacettepe University Institute of Health Sciences, Department of Stem Cell Sciences, Center for Stem Cell Research and Development, Ankara, Turkey, 06100 PKNOX2 is a member of TALE superfamily that are involved in maintaining embryonic development and tissue homeostasis (e.g. hematopoiesis). Previously, we obtained higher PKNOX2 level in bone marrow derived mesenchymal stem cells (BM-MSCs) of Fanconi anemia (FA), a disease under a high risk of developing AML. Given the crosstalk between hematopoietic stem cells (HSCs) and MSCs in BM, we hypothesized that altered PKNOX2 level may have a role in progression of FA defective haematopoiesis. To model upregulated protein level of patients, donor BM-MSCs were transfected with PKNOX2-pCMV6 by electroporation. PKNOX2 mRNA by qPCR and protein level by western blotting was assessed. Transfected cells were then cocultured with CD34+ HSCs for 4 days and their fold expansion rate, colony forming capacity (CFU) and CD34/CD38 levels (by flow cytometry) were determined. PKNOX2 overexpression was confirmed at both mRNA (fold change= 22.40) and protein level (1.18±0.08). CD34+ HSCs from PKNOX2:BM-MSC cocultures had higher fold expansion rate and higher CD38 but lower CD34 levels compared to controls. HSCs cocultured with BM-MSCs transfected with PKNOX2 had higher number of CFUs especially BFU-E than controls. In conclusion, CD34+ cells cocultured with PKNOX2:BM-MSCs have higher fold expansion rate, CD38 level and differentiation capacity. In the light of the previous studies suggesting induction of CD38 during the proliferative stage of hematopoiesis, high PKNOX2 level of FA BM-MSCs may prompt HSC proliferation and differentiation towards common myeloid progenitors via CD38 induction, thus may have an influence on triggering malignancy. This work was supported by TUBITAK project 214Z033.

P12 New role of vitamin D receptor (VDR) in the maintenance of hematopoietic and leukemic stem cells

F Zylbersztejn 1 16 E Paubelle 2 A Mupo 3 M Cheok 4 L Lieben 5 V Asnafi 9 E Macintyre 9 P Sujobert 2 J Decroocq 6 A Yokoyama 8 J Tamburini 6 V Bardet 15 S Castaigne 10 C Preudhomme 4 H Dombret 11 G Carmeliet 5 D Bouscary 6 Y Z Ginzburg 12 H De Thé 13 M Benhamou 14 R C Monteiro 14 G S Vassiliou 3 V Maguer-Satta 16 T Trovati Maciel 1 O Hermine 1 I C Moura 1 1: Imagine Institut 2: Centre Hospitalier Lyon Sud 3: Wellcome Trust Sanger Institute 4: INSERM UMR 837 5: Laboratory of Experimental Medicine and Endocrinology, KU Leuven, Belgium 6: INSERM U1016 7: Université Paris Descartes, Faculté de Médecine Sorbonne Paris Cité, Paris 8: National Cancer Center Research Institute, Chiba, 277-8577, Japan 9: Department of Biological Hematology, Assistance Publique-Hôpitaux de Paris, Hôpital Necker 10: Department of Hematology, Hôpital Mignot, 78150, Le Chesnay, France 11: Hôpital Saint Louis 12: Erythropoiesis Laboratory, LFKRI, New York Blood Center, New York, NY, USA 13: Molecular virology and pathology, INSERM UMR 944, 75010, Paris 14: Center for Research on Inflammation, INSERM U1149 15: Hôpitaux Universitaires Paris-Ile de France-Ouest 16: INSERM U1052 Acute myeloid leukemia (AML) is a poor prognosis disease because of relapses due to persistence of Leukemic Stem Cells (LSC). Vitamin D (VD) is a well-known differentiating agent in AML but the role of Vitamin D receptor (VDR) remains unclear in AML. Our team has previously demonstrated that a high VD levels before treatment is a good prognostic factor in AML. To evaluate the expression of VDR in AML with normal karyotypes excluding promyelocytic AML (APL), we conducted post hoc transcriptomic analyses of data published in the literature. We found that VDR expression VDR is a new prognostic factor in AML (excluding APL) and the expression of is downstream target genes correlates with a better survival (p=0,04). Then we investigated the role of VDR in normal hematopoiesis using VDR knockout mice . These mice have an increased numbers of hematopoietic stem cells (p<0,01) with a reduced oxidative metabolism and increased quiescence. In a context of leukemogenesis, the lack of VDR induces a more immature immunophenotype and more LSC. Targeting the VDR is effective in eradicating the disease in vitro and in mouse models. Restoration of VDR pathway increases survival of Npm1c Flt3-ITD mouse model and reduces AML engraftment in secondary transplantation. Finally, we show that VDR expression is downregulated by DNA



methylation of specific CpG sites in its promoter region . These data suggest that VDR, in addition to its differentiating effect, may have a new role , and may consist in a new attractive target in AML.

P13 A glucocorticoid-dependent metabolic program supports cancer stem cell properties in breast cancer.

E NOURI 1 2 5 L Payen-Gay 1 2 5 J Guitton 1 P Jame 1 A Puisieux 1 2 5 A M Vigneron 1 2 5 1: Université Lyon 1 2: Center of Oncology 3: Inserm 4: CNRS 5: Institute of Cancer Research (ICR) Cancer cells with stemness properties - generally designated cancer stem cells (CSCs) - are at the apex of the hierarchical organisation of tumours and are believed to drive tumour recurrence and metastasis formation. However, how they perform these neoplastic activities in a nutritive environment that is distinct from the one in an established tumour is unknown. Here, we unveil the prominent role of glucocorticoid activity in the control of mammary cancer cell plasticity and the induction of metabolic pliancy necessary for the tumorigenic potential of CSCs. By regulating MAFB-dependent cell reprogramming, glucocorticoids control stemness traits in malignant epithelial cells. As an integral part of this regulation, glucocorticoids activate the hexosamine biosynthetic pathway and rewire the metabolism of CSCs. The anabolic efficiency of these cells increases then, fostering tumour and metastasis development. Together, our findings suggest that inhibition of glucocorticoid metabolic activity could be an original strategy for CSC eradication and tumour treatment.

P14 SOX9 and PML regulate glioblastoma stem cell activity

P Aldaz 1 3 I Garcia 1 S Torres 4 J Villanua 4 I Ruiz 4 N Samprón 4 A Matheu 1 1: I.I.S. Biodonostia 2: Universidad del Pais Vasco (UPV) 3: Asociación Española Contra el Cancer (AECC) 4: Hospital Universitario Donostia Glioblastoma multiforme (GBM) is the most frequent and lethal primary brain tumor, which exhibits an extremely poor prognosis. GBM contains a subpopulation of glioma stem cells (GSC) with neural stem cells characteristics that are crucial drivers of tumor initiation, recurrence and resistance to therapies. The disregulation of genes that control the role of neural stem cells seems to be a critical feature in GBM development and malignancy. Therefore, these genes could become potential therapeutic targets in the treatment of GBM. In this sense, our interest is focused on SOX9 (sex-determining region Y (SRY)-box 9 protein) and PML (promyelocytic leukemia gene), proteins which we have recently shown to be important for the metastatic features of breast cancer stem cells (Martin et al., Nature Communications 2016). Our results show that both SOX9 and PML are enriched in this population of GSCs and are important for its maintenance. Indeed, SOX9 and PML act as oncogenes in this tumor through the promotion of proliferation, self-renewal and tumor formation. Moreover, we found that PML maintains GSCs through the expression of SOX9. We also tested their clinical relevance finding that their expression is elevated in biopsies from a subset of patients with poor prognosis postulating that PML and SOX9 inhibition is a promising strategy to combat chemotherapy resistance. In line with this idea, we found that Arsenic Trioxide, a PML inhibitor, impairs GSCs activity through SOX9. Understanding the mechanism by which PML and SOX9 participate in GSC activity might facilitate the development of novel therapeutic strategies.

P15 Neural-competent cells of mouse ventral skin belong to the Schwann lineage

In the trunk dermis, dorsal precursors that are traced by Myf5 expression are supposed to be originated in the dermomyotome and generate Schwann cells, a cell type previously believed to be of exclusive neural crest origin. Derivation of neural lineage cells from non-neural tissue is uncertain from a developmental point of view. Since we had previously shown that human ventral dermis-derived precursors originate from Schwann cells, and that their neural competence was mediated by SOX2 expression levels, we wanted to test if this was also the case for mouse ventral dermis-derived Myf5+ precursors. Contrary to expectation, we here describe the existence of a Myf5+ precursor cells in ventral dermis that retains the capacity to generate neural (Schwann) derivatives. Close inspection showed that these precursor cells had an in situ localization consistent with terminal Schwann cells. As expected, SOX2 expression levels where higher in the Myf5+ lineage and in situ localization of SOX2+ cells was partially overlapping that of Myf5+ cells, indicating that neural competence of precursors of the mouse ventral dermis is also regulated by SOX2. These results shed light on the developmental origin of dermal precursors and highlight the importance of



elucidating the identity of dermal cells with neural competence prior to their pretended use in neural tissue engineering approaches.

P16 Are SOX2 expression levels involved in dermal stem cell aging?

L Yndriago 1 H Iribar 1 A Gutierrez-Rivera 1 V Perez-López 1 A Izeta 1 1: Biodonostia Institute Stem cells constitute a constant repair system for the body and their loss or dysfunction leads to tissue aging. We have previously shown that (i) human dermal stem cells lose functionality with age, becoming senescent; (ii) dermis-resident Schwann cell precursors and pericytes constitute stem cells, their stemness being mediated by SOX2 expression levels. During the dermal aging process the expression levels of SOX2 might also modulate the activity of stem cells. To test this hypothesis we characterized the aged skin stem cell niches in young vs aged C57BL/6 mouse skin. We found a reduction in subepidermal microvessels and nerve terminals that correlate with a decrease in the in vitro functionality of dermal stem cells. Concomitantly, SOX2 expression levels seem to be reduced in C57BL/6 aged skin. We then studied young haploinsufficient SOX2EGFP mice, which express lower levels of SOX2 in various tissues including the skin. However haploinsufficient dermal stem cells showed no apparent alteration in neural differentiation in vitro. To assess if skin function was affected by SOX2 haploinsufficiency, we analyzed peripheral nerve function through Hot Plate (for thermoreception) and Von Frey filament assays (for mechanoreception). In both cases a sharp reduction was detected in aged vs young mice, while genotype differences were found only in thermoreceptive response, which was diminished in SOX2EGFP mice as compared to wt littermates. Finally, histomorphological analyses showed a significant reduction in elastic fibers in SOX2EGFP mice, indicating that dermal stem cell disfunction was compromising the replenishment of the dermal compartment.

P17 Late myeloid progenitors regenerate haematopoiesis in submyeloablatively irradiated mice

K Faltusova 1 P Paral 1 M Molik 1 C L Chen 1 L Sefc 1 E Necas 1 1: Institute of Pathophysiology, 1st Medical Faculty, Charles University Regeneration of damaged hematopoiesis is thought to arise from stem cells (HSCs). We studied the hematopoiesis regeneration initiated by a small number of repopulating cells surviving in mice irradiated with 6 Gy. Two weeks after irradiation, hematopoiesis rapidly expanded. Analysis of bone marrow showed immature Lin-c Kit+ cells with decreased c Kit expression level and decreased c Kit mRNA. In normal bone marrow, the low c Kit expression level occurred in a majority of Sca-1- myeloid committed (late) progenitors. After irradiation, a fraction of Lin-c kitlow Sca 1- expressed Sca 1 antigen and were converted into Sca 1+ cells. We further analyzed Lin-c Kit+ cells in regenerating bone marrow according to the expression level of CD71 (transferrin receptor). This reveled presence of cell clusters with highly variable Sca 1/CD71 expression pattern. The cells forming distinct clusters differed in their proliferation rate. Furthermore, the analysis of the cells using anti-IL7R, anti-CD34 and anti-CD16/32 antibodies showed that distinct cell clusters contained megakaryocytic-erythroid (MEP), granulocytic-macrophage (GMP) and common myeloid progenitors (CMP) in various proportions. We also competitively transplanted regenerating bone marrow against normal bone marrow and analyzed resulting chimeric blood and bone marrow after 3 weeks. This clearly showed that while normal bone marrow produced both granulocyte/macrophages and B-cells, the regenerating bone marrow produced predominantly only granulocyte/macrophages. Our research demonstrates that the very late myeloid progenitors are activated in damaged bone marrow, expand their populations, and become the first source of red blood cells, granulocytes and platelets.

P18 Sca1 negative haematopoietic progenitor cells markedly differ in proliferation rate and are vital for blood cell production

P Paral 1 K Faltusova 1 M Molik 1 N Renesova 1 2 L Sefc 1 E Necas 1 2 1: Institute of Pathophysiology, 1st Medical Faculty, Charles University 2: BIOCEV, 1st Medical Faculty, Charles University The haematopoiesis steadily generates a large number of blood cells by intensive cell proliferation. Haematopoietic stem cells (HSCs) are very rare cells and divide infrequently, hence the production of mature blood cells stems from



the proliferation of progenitors. In the mouse, various types of progenitors were characterized, and the aim of our study was to find out to which extent these distinct populations contributed to mature haematopoiesis. Flow cytometry was used for immunophenotypic characterisation of haematopoietic stem and progenitor cells (HSPCs). The percentage of DNA-synthesizing (S-phase) HSPCs was determined by 5-bromo-2-deoxyuridine incorporation into DNA, and their apoptotic rate was detected by Ghost Dye™ Red 780 staining. We estimated the production rates of various types of HSPCs according to the percentage of S-phase cells, their number in bone marrow, and by assuming a constant duration of the S-phase of 5 hours. The S-phase fraction significantly differed in various types of HSPCs. The values ranged from ≈ 1 % to ≈ 90 % of cells in S-phase, and were a characterising feature of particular types of HSPCs. The CD71 (transferrin receptor) expression level closely correlated with the proliferation rate in all types of HSPCs. CD71 expression was the highest in the Lin-cKit+Sca1- cells committed to the megakaryocyte-erythroid development. Surprisingly, CD71 expression was also high in the cells committed to the granulocyte-macrophage development. The HSPCs lacking the Sca1 antigen generated approximately 22 times more cells than the Sca1 expressing ones.

P19 SOX9 acts with canonical WNT signaling to drive gastric cancer progression

E Carrasco-García 1 M Garcia-Puga 1 3 J C Santos 4 5 M Ribeiro 4 5 A Matheu 1 2 1: I.I.S. Biodonostia 2: IKERBAQUE Basque Foundation for Science (Bilbao,Spain) 3: Universidad del Pais Vasco 4: State University of Campinas (Sao Paulo, Brazil) 5: Universidade Sao Francisco (Sao Paulo, Brazil) Gastric cancer (GC) remains one of the leading causes of global cancer mortality due to therapy resistance, with Helicobacter pylori (H. pylori) infection being a major risk factor. In this study, we report the significance of the induction of the stem cell regulator SOX9 in H. pylori-infected human samples of gastritis and GC, paralleling increased levels of the pro-inflammatory cytokine TNFα. Moreover, SOX9 induction was more intense in specimens infected by the pathogenically significant CagA positive strains of H. pylori. Notably, we found that SOX9 was required for bacteria-induced GC cell proliferation, induction of β-catenin and acquisition of cancer stem cell-like properties. Analysis of three large clinical cohorts of patients revealed high SOX9 levels in GC with advanced tumor stage and poor outcome. Functionally, SOX9 silencing in GC cells enhanced apoptosis and senescence, concomitantly with a blockade of gastric cancer stem cells’ (gCSCs) self-renewal and reduced in vivo tumor initiating capability. Paralleling these effects, we also found SOX9 to mediate cisplatin chemoresistance associated with reduced disease-free survival. Mechanistic interactions between SOX9 and β-catenin expression suggested the existence of a regulatory role for SOX9 targeting the WNT canonical signaling pathway. Taken together, our findings establish the importance of the SOX9-β-catenin axis in gastric cancer pathobiology, with implications in gCSCs’ maintenance and therapy resistance. As a consequence, targeting WNT-SOX9 signaling represents a rational therapeutic strategy in GC.

P20 Function of the thyroid hormone nuclear receptor TRα1 in the intestinal epithelium stem cells

M Godart 1 C Frau 1 C Le Nevé 1 J Nadjar 1 S Ansieau 1 M Plateroti 1 1: Cancer Research Center of Lyon Background. Thyroid hormones (THs) control several aspects of gut development and homeostasis. The paradigm is amphibian metamorphosis, where they are responsible for gut remodelling and emergence of the stem cells. In previous studies we showed that THs play a fundamental role in regulating cell proliferation during the postnatal development. From a molecular point of view, THs and their nuclear receptor TRa1 control several cell-cycle controlling genes as well as the Wnt and Notch pathways, key regulators of intestinal development and homeostasis. With the aim to get more insights into TRa1-dependent control of gut development and the balance between cell proliferation and cell differentiation of the intestinal stem/progenitor cells we are currently using the Lgr5-EGFP-CreERT2 mice crossed with TRAMI or vil-TRa1-Ind mice, which constitute respectively tamoxifen inducible TRa1 loss- and gain-of-function within the Lgr5-expressing stem cells. Results. We used 3D organoids and analysed the effects of tamoxifen treatment at different time points. Interestingly, from the first point analysed (3-day), in TRAMI/Lgr5-EGFP-CreERT2 organoids we observed decreased Wnt and Notch pathways as well as stem cell and proliferation markers expression. An opposite effect was observed in vil-TRa1-Ind/Lgr5-EGFP-CreERT2. We are currently analysing a panel of differentiation markers to evaluate an effect on lineage commitment and terminal differentiation. Conclusions and perspectives. Our results strongly indicate that modulating TRa1 expression or activity has a rapid and strong effect on the intestinal stem cells. We are currently using an approach of cytometry and Facs to analyse in detail the characteristics and potentialities of the TRa1-altered stem cells.



P21 Functional interplay between the thyroid hormone nuclear receptor TRa1 and the Wnt pathway in the induction of intestinal tumorigenesis

J Uchuya-Castillo 1 J Nadjar 1 C Le Nevé 1 S Ansieau 1 M Plateroti 1 1: Cancer Research Center of Lyon Background. Thyroid hormones are key players of intestinal development and homeostasis. Our team demonstrated their function via the nuclear receptor TRa1 in controlling the balance between proliferation and differentiation of the murine intestinal epithelial precursors through the activation of the Wnt/b-catenin pathway. Interestingly, the overexpression of TRa1 in the intestinal epithelium (vil-TRα1 mice) was sufficient to induce aberrant and hyper-proliferative crypts and eventually adenomas. In addition, TRa1 overexpression in a mutated Apc gene background (vil-TRα1/Apc+/1638N mice) was responsible for the acceleration of intestinal carcinogenesis compared to the simple Apc mutants. With the aim to gain molecular insights into the TRa1-dependent acceleration of the tumorigenic process upon Apc-gene mutation, we used an approach of comparative transcription profile. Results. We defined a molecular signature of the adenocarcinomas overexpressing TRa1 which includes several actors of the Wnt and Notch pathways, intestinal stem cell markers and genes closely correlated with cancer development and progression. Among them, Wif1 encodes for an inhibitor of the Wnt pathway and it is very frequently inactivated by hypermethylation in colorectal cancer. Interestingly Wif1 mRNA expression was highly down regulated in vil-TRα1/Apc+/1638N adenocarcinomas and the presence of a TRa1 binding site in its promoter suggests a direct transcriptional regulation. We confirmed an inverse correlation between the expression of Wif1 and TRa1 in a cohort of human and mouse tumor samples, both at mRNA and protein level. We are currently using 3D-cultures to study in more detail the regulation of Wif1 by TRa1 and the impact on tumor induction.

P22 Adult neurogenesis in prion disorders: a neuro « regenerative / degenerative » paradox

A Relano-Gines S Cosenza 2 C Le Souder 2 C Monzo 2 A Gabelle 2 S Lehmann 2 C Crozet 2 1: Inserm 2: INSERM U1183 Montpellier, IRMB During the past decade, several studies have consistently shown that neural stem cells (NSC) reside in the adult mammalian central nervous system and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle (LV) or the subgranular zone in the Dentate Gyrus (DG) of the hippocampus (H). Following brain injury, this adult neurogenesis can even be increased and it is accompanied by a migration of neural precursors towards the injured area. Adult neurogenesis might be part of the physiological regenerative process, however it might become impaired by the disease’s mechanism and therefore contribute to neurodegeneration. In prion disorders this endogenous repair system has rarely been studied. Whether adult neurogenesis plays a role or not in brain repair or in the propagation of prion pathology remains unclear. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders.

P23 The Critical Limb Ischemia Treatment using Adipose Derived Stem Cells Thepapeutic Factor Concentrate and Extracelular Matrix Microparticles

K Vitkova 1 J Jurcikova 1 L Porubová 1 V Procházka 1 L Pavliska 2 B H Johnstone 3 1: 4MEDi-Centrum bunecne terapie a diagnostiky a.s. 2: University Hospital Ostrava 3: NeuroFx,LIC The aim of this preclinical study is to test the safety and efficacy of administration of human adipose-derived stem cells (ASC-CM) individualy and in combination with Cook Injectable Small intestinal submucosa (SIS) by Cook, Biotech, Inc. in the experimental setting of the rabbit model of critical limb ischemia. Transplantation of adipose-derived stem cells (ASC) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ASC are primarily mediated through paracrine



mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) comprised of a cocktail of beneficial growth factors and cytokines. Cook Injectable SIS contains growth factors and may increase blood flow and limb salvage rates in critical ischemia patients. The ability of TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 30 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo, (2) ASC-CM, (3) SIS or (4) ASC-CM + SIS. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem at day 49 and assessed for capillary density by immunohistochemistry. Results of this study demonstrate that TFC represents a potent therapeutic combination ASC-CM and ASC-CM + SIS for patients with CLI.

P24 The effect of resveratrol on pluripotency and self-renewal of embryonic stem cells

I I Suvorova 1 B B Grigorash 1 V A Pospelov 1 1: Institute of Cytology of the Russian Academy of Sciences Due to unlimited proliferation potential and differentiation capacity embryonic stem cells(ESCs) has drawn wide attention for their potential using in replacement therapy of disease in the future. However, ESCs often display spontaneous differentiation in culture thus making the process of expanding these cells highly inefficient. Some recent studies suggest that small molecular compounds may play an important role in the maintenance of ESCs pluripotency. Resveratrol is natural polyphenol found in various plants. A series of researches evidenced that resveratrol possesses various health-benefiting effects, such as anticancer, antidiabetic, cardioprotective, neuroprotective and anti-inflammatory effects. Besides, resveratrol has been reported to demonstrate the beneficial effects for mammalian reproduction and to improve the efficiency of reprogramming somatic cells into iPSCs. However limited information is available on the effect of resveratrol in regulating self-renewal and proliferation of ESCs. The present study is aimed at studying the effect of resveratrol on pluripotency and self-renewal of mouse ESCs as well as related mechanisms. The work was supported by RFBR research project № 16-04-01562.

P25 Production of Therapeutic Factor Concentrate for Animal Study for Treating of Critical Limb Ischemia

L Porubova 1 J Jurcíková 1 2 V Procházka 1 2 K Vítková 1 2 B H Johnstone 3 1: 4MEDi - Centre for Cell Therapy and Diagnostics Inc. 2: University Hospital Ostrava 3: NeuroFX, Inc Transplantation of human mesenchymal stem cells has become powerful tool in regenerative medicine. Conditioned medium (CM) is medium harvested from cells culture containing various types of cytokines and growth factors which are secreted by the cells cultivated in the medium. This CM with secreted factors is concentrated to the therapeutic factor concetrate (TFC). For preparing of TFC mesenchymal stem cells (MSC) derived from adipose tissue were used, which possess many phenotypic and functional similarities to bone marow-derived MSC, but can be isolated from fat tissue in a relatively high volume using minimally invasive techniques. TFC was analysed for many angiogenic and inflammatory cytokines and growth factors. Levels of these factors were compared to human serum. TFC contained high levels of pro-survival and potent angiogenic factor VEGF and anti-inflamatory cytokine IL-6. These factors have the potential to stimulate endogenous repair processes, modulate inflammation and promote survival of risk tissues. The results confirmed the suitable use of TFC in regenerative medicine, e.g. treating of critical limb ischemia and foot ulcers. TFC for animal study was prepared in laboratories in Park 4MEDi - Biotech Business Park for Medicinal Innovations, located in Ostrava. 4MEDi is biomedical translational center for biomedical research and development of new processes and medicinal products in the field of Advanced Therapy. Acknowledgment: This work was supported by the Ministry of Education, Youth and Sports, Czech Republic, GESHER/MOST Program, project no. LJ14003

P26 Characterization of muscle progenitors expressing Aldehyde dehydrogenase

J ETIENNE 1 S RIVERON 1 C CATELAIN 1 J T VILQUIN 1 1: Sorbonne Universités UPMC Univ Paris 06, Inserm UMRS974, CNRS FRE3617, Myology Research Center, Association Institute of Myology, Pitié Salpêtrière Hospital, Paris, France Aldehyde dehydrogenase (ALDH) constitute a superfamily of 19 human genes and 15 murine counterparts. Their main function is to detoxify aldehydes present in cells by oxidizing them into the corresponding carboxylic acid. Moreover,



several members of this family have other roles, such as cell signalling, cell proliferation and cell differentiation through their enzymatic activity. ALDH activity can be used to isolate stem cells of various tissues (neural, hematopoietic, …). ALDH is also considered a marker of cancer stem cells in several pathological contexts. Our team first described progenitors with ALDH activity in skeletal muscle, using flow cytometry and a commercial fluorescent substrate of ALDH called Aldefluor. We observed that this population is constant through ageing, however we noted a three-fold increase in the number of these cells in human biopsies from patients suffering from Duchenne Muscular Dystrophy (DMD). Nevertheless, this increase is associated with poor muscle differentiation capacities as they also express CD34, a marker we previously found to be associated with adipogenic and chondrogenic progenitors. Furthermore, we also observed the correlation between some ALDH isoenzymes and extracellular markers, as ALDH2 and ALDH8A1 were expressed in CD34+ cells, and ALDH7A1 and ALDH3B1 were expressed in CD56+ cells. Altogether, these results suggest specific expression of ALDH by various progenitor cells isolated from muscle and potential use of ALDH as markers to follow disease progression.

P27 Esrrb is the major regulator of the G1/S-transition cyclin-E1 gene in mouse embryonic stem cells.

F GONNOT 1 2 3 P SAVATIER 1 2 3 1: Université Lyon 1 2: INSERM U1208 3: Stem Cell and Brain Research Institute Mouse embryonic stem cells (mESCs) display an unorthodox cell cycle characterised by the lack of a functional Rb pathway and robust expression of cyclin E during all cell cycle phases. Therefore, mESCs are constitutively primed for DNA replication. To understand how cyclin E, a key regulator of the G1-to-S phase transition, is regulated in mESCs, we analysed the transcriptional regulation of Ccne1 by transcription factors of the naive pluripotency network. We observed that Esrrb, Klf4 and Tfcp2l1 bound the Ccne1 promoter region on multiple sites between 0 and 1kb upstream transcription start site. Disrupting the binding sites reduced or abolished transcriptional activity in a luciferase assay. Moreover, the doxycyclin-inducible expression of Essrb, Klf4 and Tfcp2l1 up-regulated the Ccne1 mRNA level. Taken together, these results strongly suggest that Essrb, Klf4 and Tfcp2l1 control Cyclin E expression and highlight a direct connection between the naïve pluripotency network and regulation of the mitotic cycle in mESCs. We used the FUCCI reporter system to study cell-cycle dependent expression of the transcription factors that form the naïve pluripotency network. Esrrb, Klf4, Tfcp2l1 and Nanog expression oscillated during the cell cycle with a down-regulated expression between the early G1-phase and the beginning of S-phase, and then up-regulated expression between the beginning of S-phase and the G2/M-phase. These results suggest that the naive pluripotency network is destabilized transiently during the transition from the G1-phase to the S-phase of the cell cycle.

P28 The role of Adamts18 in epithelial-basal membrane interactions that control the stem cell function downstream of progesterone receptor signaling in the mammary gland development

D Ataca 1 S Cagnet 1 C Brisken 1 1: École Polytechnique Fédérale de Lausanne The female sex hormone progesterone controls mammary gland development. This involves extensive changes in the extracellular matrix (ECM) and the stroma, the molecular basis of which remains largely unexplored. ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are extracellular, multidomain enzymes. We identified ADAMTS18 is downstream as a target gene of progesterone and show that it is expressed in myoepithelial/basal cells, which interact with the basal lamina. The substrates of ADAMTS18 are unknown but other family members are implicated in the processing of ECM proteins such as fibrillar collagens and proteoglycans like aggrecan, versican. To gain insights into the in vivo functions of this secreted protease, we generated mice deficient for ADAMTS18. The mutant mice have delayed ductal elongation and decreased side branching in adulthood. This defect is intrinsic to the mammary epithelium. Serial transplantation assays show that Adamts18 -/- mammary epithelial cells have decreased regeneration potential. In vitro colony formation assay showed that WT adult mammary glands yielded significantly more colonies than the population from Adamts18 -/-. Both results demonstrate that the defects in ductal morphogenesis in Adamts18 deficient mice may result from impaired stem cell function. The secreted ADAMTS18 might activate and trigger signaling in the cells that are part of the stem cell niche and elicit gene expression changes there.



P29 Identification of the role of the BMP pathway in Acute Myeloid Leukemia (AML) through the correlation with ΔNp73 and NANOG, themselves associated with Leukemic stem cells.

M Flores 1 T Voeltzel 1 S Jeanpierre 1 F Louache 3 X Thomas 4 F E Nicolini 1 4 C Caron de Fromentel 1 V Maguer-Satta 1: Cancer Research Center of Lyon 2: INSERM U1052 3: Inserm 4: Hospices Civils de Lyon Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy which involves the clonal expansion of myeloblasts in the bone marrow and peripheral blood. Alteration of myeloid cells results in accumulation of immature and non-functional cells. Despite the achievement of an apparent remission, the majority of patients relapse facing a poor prognosis. The presence of leukemic stem cells (LSC) may partly be responsible for these relapses. On the other hand, the role of the BMP (Bone Morphogenetic Proteins) pathway in stem cell regulation properties in several systems and the deregulation of its elements in cancer suggest the involvement of the pathway in LSC and resistance. Our team has also recently shown that BMP pathway deregulation is associated with Chronic Myeloid Leukemia development. In the present work, we identified deregulations of the BMP pathway in AML blood samples at diagnosis from patients of. Especially, we could observe correlated expressions of BMP receptor and targets with the expression of that we had previously identified as markers of the presence of Leukemic stem cells and potentially, poor prognosis. Indeed, ΔNp73 and NANOG expressions are correlated and associated with LSC features such as high proportion of LTC-IC and ALDH activity. Furthermore, we observed that low expression of ΔNp73 in AML blood samples at diagnosis is associated with better outcome. Using patient samples, KG1A cells and BMP pathway regulators, we are deciphering the links between the BMP pathway, ΔNp73 and NANOG and Leukemic stem cells and finally AML patients’ outcome.

P30 Spindle cell asymmetry: a conserved mechanism regulating asymmetric division in mouse and primate cortical precursors

D Delaunay 1 2 V Cortay 1 2 D Patti 1 2 K Knoblauch 1 2 C Dehay 1 2 1: Inserm 2: Université de Lyon I Asymmetric cell division (ACD), whereby a progenitor gives rise to two dissimilar daughter cells, is tightly regulated during corticogenesis. Although several mechanisms are known to be involved in ACD, their relative contributions remains incompletely understood. Using apical precursors live-cell imaging, we uncovered a novel form of ACD related mechanism in the mouse neocortex: the spindle shape asymmetry (SSA). SSA occurs at metaphase, is maintained during late mitosis stages and is responsible for the genesis of late born neurons. Under the control of Wnts —relayed at the cell cortex by the action of P-ERM proteins— this mechanism also operates in monkey. Taking advantage of in-vitro micropatterns and in-vivo biphotonic microscopy, we transferred our analysis to the monkey germinal zones (SVZ and OSVZ) and reported a conserved link between SSA and ACD. Altogether, SSA appear as a key player in the tight spatiotemporal control of self-renewal and differentiation in the ventricular zone and is essential to promote diversity.

P31 Identification of factors safeguarding cellular identity during oncogenic and pluripotent reprogramming

A Huyghe 1 G Furlan 1 D Ozmadenci 1 P Wajda 1 P Mehlen 1 A Puisieux 1 F Lavial 1 1: Cancer Research Center of Lyon Cellular identity and cellular plasticity are key features of development and cancer. A key challenge for developing organisms is indeed to establish the identity of the cells by restricting progressively their plasticity. In contrast, oncogenic development often implies a partial loss of cellular identity and the reacquisition of developmental programs by cancer cells. Even if these features emerged recently as fundamental steps in cancer initiation and progression, the molecular mechanisms protecting somatic cells from the conversion to a tumorigenic state remain poorly elucidated. Our lab proposes to consider the early stage of oncogenic (OR) and pluripotent (PR) reprogramming as novel models



to identify factors safeguarding cellular identity and constraining therefore induced pluripotent stem (iPS) cells generation and tumorigenesis. Even if OR and PR drive the emergence of different cell types (cancer and iPS cells respectively), the initial steps of both reprogramming scenarios tend at rendering somatic cells tolerant to a profound epigenetic rewiring while resistant to cell death and senescence. For this reason, the dissection of the early stage of both OR and PR might improve our understanding of cancer emergence. We developed large-scale approaches and original genetic models to dissect the early molecular events triggering both OR and PR (“Pluri-Onco” model).

P32 Can Epigenetic Modifiers Advance the Stem Cell Differentiation?

Epigenetic modifiers are those chemical able to affect the epigenetic signature of the genome and consequently the gene expression. Historically, an additive role for various modifiers was suggested to affect stem cell differentiation. The logic behind was rendering the genome more active and more responsive to the culturing conditions. We showed earlier that different modifiers have certain preference to activate specific lineages of differentiation. Our studies in monolayer and three dimensional pellet culture showed that the DNA demethylating agent (5-Aza-dC) can activate the osteogenic differentiation while the histone deacetylases inhibitor, Trichostatin A, induced the chondrogenic differentiation. The combination of the two agents had no additive effects. Many authors tried, through microarray approach, to investigate the gene expression profile after the administration of these agents. The overall result was that not all genes are susceptible to the agents’ effect. Our recent data suggested that certain transcription pathways were more susceptible for each of the modifiers. Extending our studies to the adipogenic differentiation, 5-Aza-dC and the histone deacetylase inhibitor ‘Suberoylanilide hydroxamic acid’ showed opposing effect. The next generation sequencing showed activation of different pathways, even being under the same culturing condition. This presentation will include a general review on the effect of these modifiers, their role in stem cell differentiation based on our recent findings as well as their potential use as an additive component in tissue regeneration programs.

P33 Characterization of alternative splicing signatures of neurospheres deriving from human glioblastoma xenografts

N Berabez 1 3 P Désormeaux 1 C Schmitt 4 A Idbaih 4 D Meyronet 1 2 3 F Ducray 1 2 3 M Gabut 1 3 1: Cancer Research Centre of Lyon (CRCL, UMR Inserm 1052 CNRS 5286), Centre Léon Bérard, Lyon, France 2: Department of Neurology, Hôpital Pierre Wertheimer, Hospices civils de Lyon, Lyon, France 3: University Claude Bernard Lyon 1 (UCBL), Lyon, France 4: Brain and Spine Institute, Hôpital Pitié Salpêtrière, Paris, France Glioblastomas (GBM) are the most aggressive tumors in the adult brain. They contain a small number of treatment-resistant cells, the Glioma Stem Cells (GSCs), which are responsible for tumor initiation and relapse after standard treatments. Understanding the underlying mechanisms is needed to develop efficient therapies. Alternative splicing is a major driver of transcriptome diversity and splicing alterations are known to promote tumorigenesis. As it remains unclear to what extent alternative splicing regulation favors GBM, we propose to investigate the alterations of splicing programs in GSC in vitro models. We succeeded in maintaining several glioblastoma xenograft derived cells in non-adherent conditions as neurospheres. Neurospheres are 3D structures deriving from undifferentiated cells able to self-renew and to give rise to different types of cells. As they are enriched for stem cells, they represent one of the main models to study glioblastoma. But they remain highly heterogeneous like glioblastoma tumours hence the need to accurately characterize them. Combining FACS, qPCR (standard and microfluidic) and RNA-Seq, we aim at characterizing the molecular signatures of neurospheres deriving from tumors from different patients, at the transcriptomic and protein levels. Consistent with the literature, our data show an important inter-tumoral heterogeneity for selected stem and differentiated markers’ expression. This study aims at identifying splicing programs (qualitative changes) and gene signatures (quantitative changes) enriched in GSC comparing the transcriptomes of GSC, neural stem cells, and in vitro differentiated samples. Potential new targets emerging from this work will be considered to improve the clinical outcome of patients.

P34 Targeted release of transcription factors for cell reprogramming by a natural micro-syringe

L Berthoin 2 B Toussaint 2 F Garban 1 2 A Le Gouellec 2 B Caulier 1 2 B Polack 2 D Laurin 1 2 1: EFS Rhône-Alpes-Auvergne 2: UMR 5525 CNRS



Ectopic expression of defined transcription factors (TFs) for cell fate handling has proven high potential interest in reprogramming differentiated cells for in vitro and in vivo developments. Pluripotency or transdifferentiation induction as TF mediated differentiation is commonly produced by transfer of genetic information. The direct delivery of proteins could represent an interesting alternative but still needs significant advances to be efficient. We have successfully developed the direct delivery of proteins by an attenuated bacterium with a type 3 secretion system that does not require challenging and laborious steps for production and purification of recombinant molecules. This natural micro-syringe is able to inject TFs to primary human fibroblasts and cord blood CD34+ hematopoietic stem cells. The signal sequence for vectorization of the TF Oct4 has no effect on DNA binding to its nucleic target. One hour after injection, vectorized TFs are detectable in the nucleus. The injection process is associated with toxicity due to TFs, but not to the injection. The bacteria can be completely removed from cell cultures by antibiotics. A three days targeted release of Oct4 or Sox2 embryonic TFs results in the expression of target genes in fibroblasts and CD34+ hematopoietic stem cells. This microsyringe vectorization represents a new strategy for TF delivery and has potential applications for cell fate reprogramming. The easy molecular cloning and the direct injection without protein purification allow using this vector for any TF, including engineered versions with deletions or insertions.

P35 Deciphering deubiquitinase Usp9x function(s) in embryonic stem cells

M de Dieuleveult 1 2 B Miotto 1 1: Institut Cochin 2: FRM Self-renewal and differentiation in any cell types (called pluripotency) are the unique features harbored by embryonic stem cells (Young et al. 2011). While the transcriptional regulation of stem cell pluripotency has been extensively studied, only a limited number of studies have addressed the roles of post-translational modifications in this process. Several studies have proposed that the deubiquitinase Usp9X is a stem-cell marker based on its mRNA expression profile (Blanpain et al. 2004) and that it contributes significantly to the regulation of the core pluripotency network composed of transcription factors Nanog, Oct4, Sox2 and Sall4 (van den Berg et al. 2010). However, the exact contribution of Usp9x to ES pluripotency remains elusive. Here we report our analysis of embryonic stem cells knock down (KD) for Usp9x, and mutant for specific domains of Usp9x. Results from transcriptomic and proteomic studies indicate the role of Usp9x on gene expression and protein turn-over in ESC. We will also report the role of Usp9x on ESC renewal and the pluripotency network, as well as on ESC differentiation. Finally, we will show that a portion of the N-terminal part of Usp9x plays an important role on its function in ESC. Altogether, we will present a detailed analysis of Usp9x function in embryonic stem cells.

P36 Role of a Ribosome biogenesis protein in mouse embryonic stem cells maintenance

M Bruelle 1 3 A C Duc 1 F Bourdelais 1 L Guichard 1 F Catez 1 J J Diaz 1 F Ducray 1 2 M Gabut 1 1: Cancer Research Centre of Lyon (CRCL, UMR Inserm 1052 CNRS 5286), Centre Léon Bérard, Lyon, France 2: Department of Neurology, Hôpital Pierre Wertheimer, Hospices civils de Lyon, Lyon, France 3: University Claude Bernard Lyon 1 (UCBL), Lyon, France Control of gene expression programs orchestrating embryogenesis and early development is an active field of research. Progression in the understanding of these mechanisms could contribute to progress in regenerative medicine, pathologies modeling and oncology. Indeed, embryonic stem cells (ESC) and cancer stem cells (CSC) share properties like self-renewal (the ability to proliferate in a same state) and acute cell plasticity (pluripotency and multipotency, respectively). Some teams have thus demonstrated shared gene expression signatures between ESC and CSC in different models. Understanding the mechanisms governing ESC maintenance present thus a great potential for identifying innovative therapeutic strategies in oncology. ESC identity and regulation of gene expression programs is controlled at different level: epigenetic, transcriptionnal and post-transcriptionnal level. More recently, the role of the translationnal machinery, ribosomes, has emerged as implicated in stem cell homeostasis in different species. Starting the analysis from transcriptomic data (RNA-seq, GEO database), my team has identified different ribosome-associated proteins (RaPs) significantly enriched in mouse embryonic stem cells compared to differentiated murine cell lines. Among these candidates, we have focused on one particular RaPs which expression profile suggests specific role during differentiation: enrichment at the transcriptionnal and protein level in ESC and important decrease during differentiation. We are currently defining the function of our candidate protein in mouse ESC



maintenance (high cell proliferation potential, self-renewal and pluripotency). Moreover, this RaP being also expressed in human ESC and in some glioblastoma models, we will investigate its function in this pathological context.

P37 DeltaNp73 isoforms are involved in the immature phenotype of liver cancer cells

P Gifu 1 2 F Pez 1 2 L Bian 1 2 L Lefraçois 1 2 P Merle 1 2 C Caron de Fromentel 1 2 1: INSERM U1052-CNRS5286 2: Claude Bernard University Lyon1

Hepatocellular carcinoma (HCC) is the third cause of cancer death worldwide. Cancer stem cells (CSCs) in HCC tumours could be responsible for cancer relapse and radio-chemotherapeutic resistance. The p53 family (p53, p63 and p73) plays an important role in carcinogenesis and cell stemness. These genes encode full-length and truncated isoforms which play a role in stemness and exert tumor suppressor activity (TA isoforms) or oncogenic activity (DeltaN isoforms). We analyzed p73 expression in a cohort of human HCC. We observed that the acquired expression of DeltaNp73 truncated isoforms strongly correlated with that of the stemness factor Nanog. This result suggests that DeltaNp73 could participate to the emergence and/or the maintenance of cancer cells with immature properties (CSCs). CSCs may originate either from a mature hepatocyte or a liver progenitor cell. To characterize the role of DeltaNp73 in CSC generation, we ectopically expressed DeltaNp73 both in human liver progenitor cells and primary hepatocytes. As expected, DeltaNp73 overexpression was not sufficient per se to transform these liver cells. Nevertheless, we observed an altered differentiation. This effect on differentiation was also found in HCC cell lines. Indeed, DeltaNp73 overexpression results in an increased expression of Nanog and EpCAM, markers of progenitor liver cells. Moreover, DeltaNp73-overexpressing cells form more “hepatospheres” and bigger colonies (clonogenic assay) than the control. Taken together, our results indicate that DeltaNp73 may counteract liver cells differentiation and favor the emergence of immature cancer cells. Further experiments should be done to identify which pathways are involved in this phenomenon.

P38 Comparison of clonogenic potential of Lin-cKit+ cells from normal and regenerating bone marrow

T Heizer 1 2 F Savvulidi 1 M Molik 1 K Faltusova 1 E Necas 1 2 1: Institute of Pathophysiology, 1st Medical Faculty, Charles University 2: BIOCEV, 1st Medical Faculty, Charles University

When co-transplanted, normal bone marrow outcompetes regenerating bone marrow significantly. The striking difference is expressed even when the outcome of such competitive transplantation is corrected for transplanted number of immature Lin-cKit+ (LK) cells. We asked whether the markedly different efficiency of LK cells of normal and regenerating bone marrow origin, expressed in transplantation-based assays, would also be expressed in in vitro clonogenic assays. We examined normal bone marrow, regenerating bone marrow collected two weeks after bone marrow transplantation (A), and regenerating bone marrow obtained from submyeloablatively irradiated mice (B). Total bone marrow cells (analysed for presence of LK cells) or sorted LK cells, were cultured in MethoCult™ GF M3434, SF M3436 and M3334 media (Stem Cell Technologies, Canada). We also compared the clonogenic potential of the LK cells with high and low cKit expression level. Clonogenic potential of cKit(high) LK cells was significantly higher than that of cKit(low) cells. The LK cells derived from transplanted normal bone marrow (A) were similarly efficient to the LK cells from normal bone marrow in generation colonies of haematopoietic cells. The LK cells from the bone marrow spontaneously regenerating in submyeloablatively irradiated mice (B) were approximately twice less efficient in colony formation as compared to normal bone marrow and the regenerating bone marrow A. The low efficiency of LK cells from regenerating bone marrow in competitive transplantation-based assays thus appears to be connected with the transplantation procedure and is absent or only partially expressed in in vitro based clonogenic assays.

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