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

Em estudos anteriores desenvolvidos por este grupo de pesquisa uma expressão mais elevada de alvos transcricionais e componentes da via NF-kB, bem como altos níveis de NOTCH1, foi identificada em células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) quando comparadas às CTH CD34+ de medula óssea (MO). Este grupo verificou ainda, por comparação das células CD34+ com as CD133+ (mais primitivas) que diversos fatores de transcrição (FT) envolvidos com o potencial de hemangioblasto, com a autorenovação das CTH, e com a diferenciação linfóide; como: RUNX1/AML1, GATA3, USF1, TAL1/SCL, HOXA9 e HOXB4 apresentaram-se mais expressos em células mais primitivas. A potencial participação das vias Notch e NF-kB na regulação destes FT tem importância conceitual e prática no entendimento da biologia das CTH, e dos processos envolvidos na diferenciação destas células. Com isto em vista, este projeto teve como objetivo, estudar o papel da via NF-kB e da via Notch na regulação destes FT. Para isso, um modelo experimental in vitro, de diferenciação de CTH CD34+ em linfócitos T, foi utilizado e a influência de fatores agonistas e inibidores farmacológicos destas vias, foram avaliados por citometria de fluxo e PCR em tempo real. Nossos resultados evidenciam o papel da via Notch na regulação transcricional de HOXB4 e GATA3 em células-tronco hematopoéticas CD34+ humanas, o que foi confirmado com base na expressão dos alvos diretos de Notch (HEY1 e HES1). Notamos ainda, que a expressão dos transcritos HES1, GATA3 e HOXB4 é prejudicada pela síntese protéica das CTH, uma vez que quando empregamos o prétratamento com a droga CHX há aumento da transcrição dos mesmos. Também podemos inferir que a ação do TNF- é positiva sobre esses transcritos, já que quando o utilizamos há elevação do nível de expressão desses transcritos, com exceção a HES1. Em relação ao cocultivo das CTH com as células estromais de camundongos, verificamos que apenas a linhagem OP9-DL1 detém a capacidade de promover a diferenciação celular T, e isso foi comprovado pelo surgimento de células comprometidas com a linhagem linfocítica T, através da presença dos marcadores de superfície específico CD7+ e CD1a+. Esses resultados auxiliarão na compreensão dos mecanismos moleculares de regulação transcricional envolvidos não apenas na diferenciação de linfócitos T, mas também na manutenção de um estado mais primitivo das CTH. Este conhecimento pode vir a contribuir com o desenvolvimento ou otimização de protocolos laboratoriais visando à expansão de CTH ou geração de células T para usos terapêuticos. / In previous studies by this research group a higher expression of transcriptional targets and components via NF-kB, as well as high levels of NOTCH1, was identified in hematopoietic stem cells (HSC) CD34 + cells from umbilical cord blood (UCB) compared to CD34 + hematopoietic stem cells from bone marrow (BM). This group also found, by comparing the CD34 + cells with CD133 + (more primitive) that several transcription factors (TF) involved in the potential of hemangioblast, with self-renewal of hematopoietic stem cells and to differentiated lymphocytic; as Runx1 / AML1, GATA3, USF1, TAL1/SCL, HOXB4 and HOXA9 were more expressed in more primitive cells. The potential involvement of Notch signaling pathways and NF-kB in the regulation of FT has conceptual and practical importance in understanding the biology of HSC, and the processes involved in differentiation of these cells. With this in mind, this project aimed to study the role of NF-kB pathway and Notch signaling in the regulation of FT. For this, an experimental model in vitro differentiation of CD34 + hematopoietic stem cells into T lymphocytes, was used and the influence of pharmacological agonists and inhibitors of these pathways were evaluated by flow cytometry and real-time PCR. Our results highlight the role of Notch signaling in the transcriptional regulation of GATA3 and HOXB4 in hematopoietic stem cells CD34 + human, which was confirmed based on the expression of direct targets of Notch (HES1 and HEY1). We also note that the expression of transcripts HES1, GATA3 and HOXB4 protein synthesis is hampered by the HSC, since when we use the pre-treatment with the drug there CHX increased transcription thereof. We can also infer that the action of TNF- is positive about these transcripts, since when we use it for raising the level of expression of these transcripts, except the HES1. In relation to the HSC coculture with stromal cells of mice, we found that only the line-DL1 Op9 has the ability to promote T cell differentiation, and this was evidenced by the appearance of cells committed to the T lymphocyte lineage, through the presence of specific surface markers CD7 + and CD1a +. These results will help understand the molecular mechanisms of transcriptional regulation involved not only in the differentiation of T lymphocytes, but also in maintaining a more primitive state of HSC. This knowledge may contribute to the development or optimization of laboratory protocols aimed at the expansion of HSC or generation of T cells for therapeutic use.

O Vírus Sincicial Respiratório (RSV) é reconhecido como agente causador de infecção nosocomial entre receptores de pacientes de células-tronco hematopoéticas causando morbidade e mortalidade consideráveis nesses pacientes. O objetivo desse estudo foi caracterizar as cepas do RSV isoladas de receptores de transplante de células-tronco hematopoéticas (TCTH) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo durante sua estação. As cepas do RSV foram tipadas (em grupo A ou B) e genotipadas. Das sete cepas analisadas dos receptores de TCTH durante o ano de 2001, somente duas pertenciam ao grupo B, as outras cinco eram pertencentes ao grupo A. Dessas sete cepas, três eram altamente relacionadas e haviam infectados pacientes que freqüentavam o ambulatório. Em 2002, das doze cepas analisadas, três pertenciam ao grupo A e as outras nove pertenciam ao grupo B. Sete cepas eram altamente relacionadas entre elas e eram também de pacientes de ambulatório sugerindo que a transmissão em hospital-dia era mais provável. Enfim, múltiplços genótipos do RSV co-circularam nas unidades de TCTH (ambulatório e enfermaria) do Hospital das Clínicas entre 2001 e 2002. A transmissão nosocomial foi mais provável ocorrer no ambulatório da unidade de TCTH quando comparada à enfermaria. Políticas de controle de infecção devem ser também implementadas em ambulatórios para evitar transmissão nosocomial do RSV e outros vírus respiratórios em pacientes de ambulatório. / Respiratory Syncytial Virus is recognized as the leading cause of nosocomial respiratory infection among recipients of hematopoietic stem cell transplant (HSCT) causing considerable morbity and mortality among theses patients. The aim this study was characterize the strains of Respiratory Syncytial Virus in recipients Hematopoietic Stem Cell Transplant Unit (HSCT) at Hospital das Clínicas, University of São Paulo Medical School during RSV season in symptomatic HSCT recipients at Hospital das Clínicas. The strains of RSV was typed (in group A or B) and genotyped. Of the seven strains analyzed from HSCT recipients during 2001, only two belonged to group B, the other five belonged to group A. Of these seven strains, three were closely related and were from outpatients. In 2002 , of the twelve strains analyzed, three belonged to group A and the other nine belonged to group B. Seven strains were closely related and were also from outpatients suggesting that nosocomial transmission in hospital-day was more likely. In conclusion, multiples genotypes of RSV co-circulated in the Hematopoietic Stem Cell Transplant units (ward and dayhospital) of Hospital das Clínicas between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT Day-hospital as compared to the HSCT ward. Infection control practices should be also implemented at Day-hospital Units to avoid nosocomial transmission of RSV and other respiratory viruses in outpatient units.

As células-tronco hematopoéticas (CTHs) e estromais mesenquimais multipotentes (CTMs) isoladas da medula óssea vêm sendo utilizadas como fonte autóloga no tratamento de doenças autoimunes, como a esclerose múltipla (EM). As CTHs dão origem a todas as células dos sistemas hematopoético e imunológico e as CTMs possuem propriedades imunomoduladoras pela liberação de fatores solúveis e interação célula-célula. Existem trabalhos que sugerem que as doenças autoimunes sejam provenientes de defeitos intrínsecos nas células-tronco precursoras da medula óssea. Com o intuito de avaliar se as CTHs e CTMs de pacientes com EM possuem alterações intrínsecas, o objetivo geral deste trabalho foi avaliar o perfil de expressão gênica diferencial por microarrays de CTHs e CTMs de pacientes com EM, além de avaliar o perfil de expressão gênica de CTMs após o transplante autólogo de CTHs e a capacidade imunomoduladora in vitro das CTMs de pacientes. As CTHs e CTMs foram isoladas da medula óssea de pacientes com EM e doadores saudáveis, após consentimento informado. As CTHs foram isoladas por colunas imunomagnéticas e as CTMs foram isoladas por gradiente de densidade e submetidas à caracterização morfológica, imunofenotípica e capacidade de diferenciação em adipócitos e osteócitos. O RNA das CTHs e CTMs foi extraído e purificado e o perfil de expressão gênica foi avaliado por microarrays, utilizando hibridações em lâminas contendo 44.000 sondas. A capacidade imunomoduladora das CTMs de pacientes e controles foi avaliada por ensaios de cocultivo com linfócitos alogênicos e as citocinas foram quantificadas no sobrenadante por CBA flex e ELISA. Este estudo foi aprovado pelo comitê de ética do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Os resultados mostraram que as CTHs de pacientes possuem perfis de expressão gênica diferentes dos controles, com 2.722 genes diferencialmente expressos, envolvidos em vias de sinalização importantes para manutenção/proliferação das CTHs e diferenciação em linhagens específicas durante a hematopoese. Dentre essas sinalizações estão incluídas as vias da apoptose, Wnt, Notch, mTOR, PI3K/Akt e Ca/NFAT, sugerindo que as CTHs de pacientes com EM possuam alterações intrínsecas que podem estar relacionadas com a patogenia da doença autoimune. As CTMs isoladas de pacientes com EM exibiram aparência senescente e reduzida expressão de marcadores imunofenotípicos. Com relação à expressão gênica, as CTMs de pacientes possuem perfil diferente das CTMs controle, sendo detectados 618 genes diferencialmente expressos, incluindo genes relacionados à sinalização FGF, HGF, sinalização de moléculas de adesão e moléculas envolvidas nos processos de imunorregulação, como IL10, IL6, TGFB1, IFNGR1, IFNGR2 e HGF. O perfil de expressão gênica das CTMs de pacientes pós-transplante assemelhou-se ao perfil das CTMs pré-transplante. Ensaios de cocultivo de CTMs com linfócitos alogênicos mostraram que as CTMs de pacientes possuem capacidade antiproliferativa reduzida em relação às CTMs controle, e ainda, secreção reduzida de TGF- e IL-10 no sobrenadante das coculturas. Esses dados sugerem que as CTMs isoladas de pacientes com EM possuam alterações fenotípicas, transcricionais e funcionais. Embasados nesses achados, concluímos que as CTHs e as CTMs de pacientes com EM possuem alterações intrínsecas que podem estar relacionadas com a patogenia da doença. Uma vez que as CTMs sejam células com grande potencial terapêutico para controle da EM em pacientes refratários aos tratamentos convencionais, as alterações encontradas sugerem que CTMs de doadores saudáveis sejam mais adequadas em aplicações clínicas. / Bone marrow hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) have been used as an autologous source to treat autoimmune diseases, such as multiple sclerosis (MS). HSC give rise to all hematopoietic and immune system cells, and MSCs exhibit immunomodulatory properties by releasing soluble factors and by cell-cell interactions. Evidence indicates that bone marrow stem cells obtained from patients with autoimmune diseases may present intrinsic defects. To assess whether or not HSC and MSC of MS patients have intrinsic defects, the main objective of this study was to evaluate the differential gene expression profiles of HSC and MSC from MS patients before and after autologous HSC transplantation, and additionally, to evaluate the in vitro immunomodulatory ability of patient MSCs. Bone marrow HSC and MSCs were isolated from MS patients and healthy donors. HSCs were isolated by immunomagnetic columns and MSCs were isolated by gradient density and cultured until the third passage. MSCs were characterized according to morphology, immunophenotypic markers and cell differentiation into adipocytes and osteocytes. HSC and MSCs mRNAs were extracted, purified, and the gene expression profile was evaluated by microarray hybridizations, using a platform containing 44.000 probes. The immunomodulatory activity of patient and control MSCs was assessed by coculture assays with allogeneic lymphocytes. Cytokines were quantified in coculture supernatants by ELISA and CBA flex. This study was approved by the Ethics Committee of the University Hospital of the School of Medicine of Ribeirão Preto. The results showed that the patient HSCs exhibited a distinctive gene expression profile when compared to healthy HSCs, yielding 2.722 differentially expressed genes, involved in essential HSC signaling pathways for maintenance, proliferation and differentiation into specific lineages during hematopoiesis. Among these signaling pathways were included, apoptosis, Wnt, Notch, mTOR, PI3K/Akt and Ca/NFAT, suggesting that patient HSCs have significant intrinsic transcriptional alterations that may be associated with MS pathogenesis. Regarding MSCs isolated from MS patients, they exhibited senescence appearance, decreased expression of immunophenotypic markers, and also exhibited a distinctive gene expression profile in relation to healthy MSCs, yielding 618 genes differentially expressed genes, included in FGF and HGF signaling pathways, adhesion molecules, and genes involved in immunoregulation processes, such as IL-10, IL-6, TGFB1, IFNGR1, IFNGR2 and HGF. Coculture assays of control or patient MSCs with allogeneic lymphocytes showed that patient cells exhibited reduced antiproliferative activity as compared with controls, and also exhibited reduced secretion of TGF- and IL-10 cytokines in coculture supernatants. These data suggest that MSCs isolated from MS patients have phenotypic, functional and transcriptional defects, highlighting genes related to MSC maintenance, adhesion and immunomodulatory effects. According to these results, we concluded that patient HSCs and MSCs have intrinsic defects that may be associated with the disease per se. Considering that MSCs exhibit great therapeutic potential to control MS patients refractory to conventional treatment, the major MSCs alterations observed in this study indicate that healthy MSCs may be more suitable for MS cell therapy.

Chez l’adulte les premières étapes du développement hématopoïétique se déroulent dans la moelle osseuse (MO). La contribution de cellules d’origine mésenchymateuse, appelées niches stromales, a été démontrée dans le cas de la maintenance des cellules souches hématopoïétiques (CSH) et du développement des lymphocytes B (LB). Ainsi la maintenance des CSH dépend de niches périvasculaires sécrétant CXCL12 et SCF. Par ailleurs les LB les plus précoces (preproB) sont en contact de cellules stromales CXCL12+, puis migrent vers des cellules stromales exprimant l’interleukine-7 lors de leur différentiation en cellules proB. L’expression du préBCR, marque ensuite l’entrée dans le stade préB. À ce stade, les cellules sont au contact de cellules stromales galectine-1+.Malgré les progrès obtenus dans la compréhension du rôle des niches stromales, leur hétérogénéité et les mécanismes contrôlant la migration et l’adhésion des cellules hématopoïétiques en différenciation restent à mieux définir. Dans cet objectif, nous avons caractérisé phénotypiquement les cellules stromales de la MO mais aussi démontré l’existence d’une niche multi-spécifique, associée aux sinusoïdes et capable de soutenir les CSH et les LB.La contribution des niches dans le développement et la résistance aux traitements des Leucémies Aigues Lymphoblastiques de type B (LAL-B), équivalents pathologiques des LB en développement, a aussi été démontrée. Au cours de mon travail de thèse nous avons révélé l'influence d'un facteur exprimé par des cellules stromales de la MO sur la prolifération des LAL-B. À terme, ces travaux permettront de développer des traitements ciblant les fonctions protectrices des niches tumorales. / In adults, the early stages of hematopoietic development take place in the bone marrow (BM). The contribution of specialized cells of mesenchymal origin, called stromal niches, has been demonstrated in the case of hematopoietic stem cell (HSC) maintenance and B lymphocyte development. Indeed, the maintenance of HSC depends on perivascular niches secreting CXCL12 and SCF. Furthermore progenitor B cells (preproB) are in contact with CXCL12+ stromal cells and migrate towards interleukin 7 expressing stromal cells during their differentiation into proB cells. PreBCR expression then marks the entrance into the preB cell stage. At this point, the cells are in contact with galectin-1+ stromal cells.Although progress have been made in understanding the role of stromal cell niches, their heterogeneity and the mechanisms controlling migration and adhesion of differentiating hematopoietic cells are controversial and remain to be defined. With this objective, we characterized phenotypically BM stromal cells but also demonstrated the existence of a multi-specific niche, associated to sinusoids and able to support both HSC and early B cells.The contribution of BM niches in the development and resistance to treatment of B cell Acute Lymphoblastic Leukemia (B-ALL), pathological equivalent of developing B cells has also been demonstrated. During my PhD, our work revealed the influence of a factor expressed by BM stromal cells on the proliferation of B-ALL. Ultimately, this work will allow the development of treatments targeting the protective functions of tumor niches.

During embryonic development a variety of tissues and organs such as the lung, eye, and kidney are being formed. The generation of functional organs is regulated by reciprocal cell-cell interactions. Via the secretion of soluble molecules one type of cells affect the fate of their neighboring cells. A central issue in organogenesis is how a cell interprets such extrinsic signals and adopts a specific fate, and how the cell in response to this signal establishes reciprocal signaling. Transcription factors play a critical role in this process and my thesis focuses on the role of the LIM-homeodomain transcription factor, Lhx2, in the development of three different organ systems, the liver, the hematopoietic system and the olfactory system. The liver is formed from endoderm of the ventral foregut and mesenchyme of the septum transversum (st) and its development depends upon signaling interactions between these two tissues. As the liver becomes a distinct organ it is colonized by hematopoietic cells and serves as hematopoietic organ until birth. The fetal liver provides a microenvironment that supports the expansion of the entire hematopoietic system (HS) including the hematopoietic stem cells (HSCs). Liver development in Lhx2-/- embryos is disrupted leading to a lethal anemia due to insufficient support of hematopoiesis. To further investigate the role of Lhx2 in liver development I analyzed gene expression from the Lhx2 locus during liver development in wild-type and Lhx2-/- mice. Lhx2 is expressed in the liver associated st mesenchymal cells that become integrated in the liver and contribute to a subpopulation of hepatic stellate cells in adult liver. Lhx2 is not required for the formation of these mesenchymal cells, suggesting that the phenotype in Lhx2-/- livers is due to the presence of defective mesenchymal cells. The putative role of Lhx2 in the expansion of the HS was examined by introducing Lhx2 cDNA into embryonic stem cells differentiated in vitro. This approach allowed for the generation of immortalized multipotent hematopoietic progenitor cell (HPC) lines that share many characteristics with normal HSCs. The Lhx2-dependent generation of HSC-like cell lines suggests that Lhx2 plays a role in the maintenance and/or expansion of the HS. To isolate genes putatively linked to Lhx2 function, genes differentially expressed in the HPC lines were isolated using a cDNA subtraction approach. This allowed for the identification of a few genes putatively linked to Lhx2 function, as well as several stem cell-specific genes. The antagonist of Wnt signalling, Dickkopf-1 (Dkk-1), was identified in the former group of genes as it showed a similar expression pattern in the fetal liver, as that of Lhx2 and expression of Dkk-1 in fetal liver and in HPC lines appeared to be regulated by Lhx2. This suggests that Dkk-1 plays a role in liver development and/or HSC physiology during embryonic development. During development of the olfactory epithelium (OE) neuronal progenitors differentiate into mature olfactory sensory neurons (OSNs) that are individually specified into over a thousand different subpopulations, each expressing a unique odorant receptor (OR) gene. The expression of Lhx2 in olfactory neurons suggested a potential role for Lhx2 in the development of OSNs. To address this OE from Lhx2-/- and wild-type mice was compared. In the absence of functional Lhx2 neuronal differentiation was arrested prior to onset of OR expression. Lhx2 is thus required for the development of OSN progenitors into functional, individually specified OSNs. Thus, Lhx2 trigger a variety of cellular responses in different organ systems that play important roles in organ development in vivo and stem cell expansion in vitro.

L’osteopetrosi autosomale recessiva (ARO) è un gruppo di malattie dovute a un difettoso funzionamento degli osteoclasti che preclude un rimodellamento osseo corretto. Nel 50% dei casi umani il difetto è dovuto ad una delezione nel gene Tcirg1. Il modello murino mutante oc/oc porta lo stesso difetto genetico e fenotipico umano. Nel lavoro di tesi si è dimostrato che gli epatociti fetali di 12.5 giorni di gestazione trapiantati in utero in feti mutati di 13.5 giorni di gestazione sono in grado di curare il fenotipo malato. Si è inoltre derivata una sottolinea di cellule staminali embrionali murine transgeniche per il costrutto plasmidico GOF18eGFP. Si vuole utilizzare la GFP sotto il controllo del promotore del gene Oct-4 come marcatore del livello di staminalità cellulare per microiniettare le ESC in blastocisti murine mutate oc/oc. / Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders due to defects that preclude normal function of osteoclasts. In half the cases, human ARO is due to mutations in the Tcirg1 gene. The oc/oc mutant mouse closely recapitulates human Tcirg1-dependent ARO. In ths work we demonstrate that in utero injection of allogenic fetal liver cells on 12.5 days into oc/oc fetuses at 13.5 day post coitum completely rescue the osteopetrotic phenotype. Moreover, an embryonic stem cells line transgenic for GOF18eGFP was produced. The goal is to use the GFP under the transcriptional control of the Oct-4 promoter as a marker of pluripotency of the ESC that are to microinject into oc/oc blastocysts.

No description available.

The Master´s thesis gives an overview of cord blood, its use, processing and cryopreservation. The role of cord blood as an alternative resource for transplantation is being widely discussed. In the experimental part, 50 grafts of donor cord blood were processed and the effect of cryopreservation and demographic factors of mother and child were evaluated on the cellularity of cord blood. Based on the evaluation, a recommendation about the inclusion of additional tests in routine processing of cord blood were made.

Afin d’améliorer au mieux le greffon placentaire, nous suggérons de réaliser sa culture d’expansion ex vivo dans des conditions proches de l’environnement des cellules souches hématopoïétiques in vivo. Ainsi, nous proposons que la co-culture de cellules CD34+ placentaires avec des cellules stromales mésenchymateuses (CSM) à basses concentrations d’O2 (BC-O2) pourrait contribuer à équilibrer les processus d’autorenouvellement et de différenciation afin d’obtenir un greffon optimisé. Sur le plan fonctionnel, nos résultats confirment un effet bénéfique de notre modèle expérimental par rapport aux conditions où figure la culture simple et/ou l’oxygénation atmosphérique (20%) en termes du maintien de progéniteurs (PH) primitifs (pré-CFC) et de cellules souches Scid-Repopulating Cells (SRC). Sur le plan quantitatif, l’amplification des cellules CD34+ et des PH engagés, bien qu’elle soit en retrait dans nos conditions de référence par rapport à la condition de 20% d’O2, elle demeure néanmoins importante. Par ailleurs, le rôle de l’IL-3 exogène se montre crucial à BC-O2 notamment en co-culture à 1,5% d’O2 où elle permet non seulement de préserver mais aussi d’amplifier le taux de SRC par rapport au témoin de cellules CD34+ de J0. Enfin, l’étude de la sécrétion des facteurs solubles et l’expression des marqueurs phénotypiques sur les CSM montre que l’IL-6, le VEGF et l’IL-8 sont plus sécrétés et les CD146, CD49a, CD54, CD200 et CD105 sont plus exprimés après incubation à 5% d’O2. Cependant, l’implication réelle de ces facteurs et antigènes dans l’effet paracrine et/ou de contact cellulaire direct menés par les CSM dans notre protocole requiert de nouvelles investigations / To optimize at best the hematopoietic engraftment, we suggest in this work to improve the ex vivo expansion conditions by moving them closer to physiology. Indeed, we propose to culture placental CD34+ (HSC/PH) on MSC layer in combination with LO2-C to ensure the amplification of HP together with the maintenance/expansion of HSC. Compared to the single culture and/or atmospheric oxygenation, our experimental model allows a better maintenance of primitive HP (Pre-CFC) and HSC together with a quite good amplification of total cells, CD34+ cells and committed HP despite of lower than control condition. Moreover, exogenous IL-3 shows crucial effect in co-culture at LO2-C (1.5% O2) since its addition better preserves and even increases the number of HSC compared to the CD34+ cells control from D0. We then studied the secretion of soluble factors in culture supernatants and found that IL-6, VEGF and IL-8 were present in larger quantities at LO2-C in both co-culture and MSC culture. Finally, the CD146, CD49a, CD54, CD200 and CD105 membrane antigens appear to be up-regulated in MSCs when incubated at 5% O2. However, the involvement of these factors and antigens in paracrine effect and/or direct cell to cell contact mechanisms at LO2-C requires further investigations. In conclusion, the combination of LO2-C and MSC would be promising in the field of HSC/PH grafts expansion to achieve its main objective of reducing the post-transplant cytopenia period together with maintaining the long-term graft potential