ringed sideroblasts (RARS) vii

Leukaemia inhibitory factor (LIF) Bone morphogenetic protein (BMP) Vascular endothelial growth factor (VEGF) Colony-forming unit-erythroid (CFU-E) Burst-forming unit-erythroid (BFU-E) Colony-forming unit-granulocyte, macrophage (CFU-GM) Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) Colony-forming unit-megakaryocyte (CFU-Mk) Spleen colony forming unit (CFU-S) Adventitial reticular cells (ARCs) Low-affinity nerve growth factor receptor (LNGFR) Stromal cell-derived factor-1 (SDF-1) Chemokine (C-X-C motif) ligand 12 (CXCL12) Osteoprotegerin (OPG) Rapidly self-renewing cells

TITRE DE LA THÈSE EN FRANÇAIS

ÉTUDE DU MICROENVIRONNEMENT MÉDULLAIRE ET DES COMPLEXES D'ADHÉRENCE FOCALE DANS LE MYÉLODYSPLASIES ET LÉUCÉMIES

RESUMÉ DE LA THÈSE EN FRANÇAIS

Les syndromes myélodysplasiques (SMD) sont considérés comme des maladies clonales des cellules souches hématopoïétiques (CSH). Le microenvironnement joue un rôle important par ses contacts direct avec les cellules progénitrices hématopoïétiques (CPH). Notre objectif était d'évaluer les défauts de croissance des cellules stromales mésenchymateuses (CSM) dans les MDS, d'explorer les molécules d'adhérence impliquées, et d'effectuer des corrélations avec leurs dysfonctionnements de croissance et les anomalies des CPH. Les CSM de MDS sont intrinsèquement pathologiques, montrant une baisse continue de la prolifération pendant 14 jours de culture et une capacité clonogénique réduite. Ces anomalies sont corrélés à une diminution des molécules d'adhérence CD44 et CD49e. Par ailleurs, le potentiel clonogénique des CPH est contrôlé par des mécanismes d'adhérence dépendant du stroma, CD49e pouvant être une des molécules impliquées. L'analyse en immunofluorescence des protéines d'adhérence focale (FA), paxilline et pFAK [Y 397 ], et des deux protéines régulatrices, HSP90αβ et p130CAS permet l'identification d'anomalies qualitatives et quantitatives. Une expression accrue de paxilline, pFAK et HSP90αβ et leur forte co-localisation nucléaire dans les CSM d'anémie réfractaire avec excès de blastes (AREB) sont corrélées avec un avantage prolifératif et un impact négatif sur la capacité clonogénique de CPH. Ces résultats ouvrent des possibilités intéressantes : la signalisation via les protéines FA pourrait être impliquée dans les interactions HPC-MSC ; par ailleurs, FAK étant une protéine cliente d'HSP90, les inhibiteurs d'HSP90 sont une potentielle thérapie adjuvante dans les myélodysplasies.

TITRE DE LA THÈSE EN ANGLAIS

STUDY OF MARROW MICROENVIRONMENT AND FOCAL ADHERENCES IN MYELODYSPLASTIC SYNDROMES AND LEUKEMIAS

RESUMÉ DE LA THÈSE EN ANGLAIS

Myelodysplastic syndromes (MDS) are regarded as clonal disorders of haematopoietic stem cells (HSC). Recent evidence demonstrates that stromal microenvironment, in addition to HSC defects, plays a particular role via its direct contact with haematopoietic precursor cells (HPC). This thesis aims at evaluating the putative growth deficiencies of mesenchymal stromal cells (MSC) from MDS individuals compared with normal controls, exploring their adhesion profile, assessing the adhesion process-involved molecular substrates, and establishing correlations with their growth patterns and HPC dysfunctions. Functional assays revealed that MSC from MDS are intrinsically pathological, show a continuous decline of proliferation over a 14-day culture and a reduced clonogenic capacity in the absence of signals from HPC. MSC growth defects significantly correlate with decreased CD44 and CD49e expression. Moreover, stroma-dependent adhesion mechanisms control HPC clonogenic potential and CD49e might be one of the molecules involved in this process. Qualitative and quantitative abnormalities of focal adhesion (FA) proteins paxillin and pFAK [Y 397 ] and of two regulatory proteins, HSP90αβ and p130CAS were identified via immunofluorescence analysis. Paxillin, pFAK [Y 397 ] and HSP90αβ increased expression, besides its stronger nuclear colocalization in MSC from RAEB correlates with a consistent proliferative advantage and has a negative impact on HPC clonogenic capacity. These results open interesting opportunities, e.g. HPC-to-MSC interactions involve FA proteins signalling, and, as FAK is an HSP90αβ-client protein, it may enhance the utility of HSP90αβ inhibitors as adjuvant therapy in MDS.

i me the opportunity to carry out this project in her lab, for her helpful comments on my thesis, and for her patience.

I acknowledge the financial support of the "Ligue Départementale contre le Cancer de la

Loire", and "Association Les Amis de Rémi" (France).

Thanks to my family for all the support and advice you have given me over the last few years -I could not have done it without you, and I will endeavour to see more of you now! Thanks to Constantin for his love, support, patience and understanding, for helping me put things into perspective on occasion, and for being my light at the end of the tunnel.

Finally, apologies to the many friends whom I have been ignoring, but who have nevertheless been there whenever I needed some moral support.

iii

I-1. Embryogenesis and early stem cell development

Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate in order to produce mature progeny cells, including both non-renewing progenitors and terminally differentiated effector cells.

Stem cells have been classified by their developmental potential as: totipotent (able to give rise to all embryonic and extra-embryonic cell types, such as trophectodermal cells, which give rise to the subsequent cell lineages that produce the placental tissues and components of the yolk sac), pluripotent (able to give rise to all cell types of the embryo proper), multipotent (able to give rise to a subset of cell lineages), oligopotent (able to give rise to a more restricted subset of cell lineages than multipotent stem cells), and unipotent (able to contribute only one mature cell type) (Figure 1). Pluripotent embryonic stem (ES) cells were first isolated by in vitro cell culture derived from the inner cell mass (ICM) of blastocysts [1]. In vivo, the ES cells create the three embryonic germ layers: ectoderm (believed to give rise to skin and neural lineages), mesoderm (believed to generate blood, bone, muscle, cartilage, and fat), and endoderm (believed to contribute at tissues of the respiratory and digestive tracts) [1].

Recently, a new category of cells, in-vitro-reprogrammed induced pluripotent stem cells (iPS) were generated from human somatic cells using the four transcription factors (Oct4, also called Oct3/4 or Pou5f1, SOX2, NANOG, and LIN28), which can reprogramme a somatic genome back into an embryonic epigenetic state [2], [3], [4]. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into all three primary germ lines [2]. Noteworthy is that the iPS production can be suppressed by the p53-p21 pathway [3].

The common marker of multiple stem cell populations is the high level activity of ATP binding cassette (ABC) transporter proteins, and although the biological significance of this property is unclear, it has been useful in the enrichment of stem cells from multiple tissues by virtue of their enhanced efflux of the fluorescent dye Hoechst 33342 (the so-called "side population", SP cells) [1]. In addition, other metabolic markers / dyes such as rhodamine123 (which stains mitochondria), Pyronin-Y (which stains RNA), and BAAA 1 (indicative of aldehyde dehydrogenase enzyme activity) are imprints of the functional stem cell activity [5]. Haematopoiesis is a multiple-step process, in which a relatively small population of haematopoietic stem cells (HSC) give rise, after a sequentially maturation stage, to all types of blood cells (Figure 2) [6].

Figure 2. Schematic diagram of haematopoietic maturation stages

The different cells identified have been clustered into three families according to the type of assay which served for their identification: in vivo transplantation assay or cytometry selection and Long Term Culture-Initiating Cell Assay (LTC-IC cultures) (the left panel), in vitro colony assays in methylcellulose culture system (middle panel), and morphological classification (right panel). Adapted from: Domen J., Wagers A. & Weissman I. L., Stem Cell Information 2011

The HSC is currently best characterized as a multipotent stem cell population [1].

The HSC pool represents only 0.05% of the whole bone marrow [7] and is able to maintain haematopoiesis over a lifetime based on its two fundamental features, slow division and high self-renewal rate [6]. A possible explanation for these slow division kinetics might be to reduce the risk of mutagenesis and defects during cell division [8], and it is also a prerequisite for maintaining haematopoiesis during a life-time, otherwise the stem cell pool might be rapidly depleted [6].

The phenotype recognized for the HSC highly enriched mouse population, which includes all multipotent progenitors, is Thy-1.1 low Sca-1 hi Lineage (Lin) -/low [7].

In humans, a small subset of HSC is capable of long-term repopulation upon transplantation (LT-HSC). These cells can be enriched by their immunophenotype as CD34 + CD38 -Thy-1 -Rh123 low [9], they are usually dividing very slowly, provide a multilineage haematopoietic engraftment ability [10], and the next compartment comprises short-term repopulating stem cells (ST-HSC) that sustain haematopoiesis only for a limited several-week span (i.e., 4-10 weeks) after transplantation and these may correspond to a CD34 + CD38 -phenotype [6], [7]. The progenitor cells (such as CMP) are included in the CD34 + CD38 + cell fraction. In the later stages, CD34 expression is absent and lineage specific markers are expressed [6].

Recent evidence also indicates the existence of a CD34 -HSC, which seems likely to be quiescent, capable to convert into CD34 + phenotype upon activation with 5-fluorouracil or after culture and, after transplantation, could revert into CD34 -phenotype again [9].

Recent experiments using CD34 -cord blood cells that are in the G 0 state show that these cells have more than 1000-fold potential to form GM-CFC, a 250-fold higher BFU-E forming capacity, and 600-fold higher expansion potential compared to G 1 cells [9], [11]. Moreover, these most primitive HSC show marrow repopulating ability, resistance of 5-FU, and low retention of rhodamine-123 (Rh123) [9], [12].

The dual function of self-renewal and differentiation is regulated by asymmetric cell divisions, where one daughter cell retains the stem cell function, whereas the other differentiating cells become a faster proliferating precursor cell [6], [13]. Alternatively, cells can undergo symmetric divisions to produce either two identical, self-renewing cells, or two differentiated daughter cells [6], [13]. The pool of HSC and progenitor cells is regulated by a feedback mechanism that is related to the number of mature cells in the blood (Figure 3) [6].

Figure 3. Self-renewal and differentiation in human haematopoiesis

Haematopoietic differentiation is a multiple-step process. A small group of long term repopulating haematopoietic stem cells (LT-HSC) replicates very slowly. The downstream compartments are increasingly committed to a specific lineage and have a fate-rate replicating capacity. Some of the progeny have to self-renew to keep the pool of haematopoietic stem and progenitor cells. The percentages of self-renewal versus differentiation are regulated by a feedback mechanism that is related to the number of mature cells in the blood (A). The stem cells can divide by either asymmetric or symmetric modes of division, and the balance between these two modes is controlled to produce appropriate numbers of stem cells and differentiated daughters (B). SP: side-population, 5-FU: 5-fluorouracil, DR: class II human leukocyte antigens molecules, Rh123: rhodamine-123 MPC: multipotent progenitor cells, CPC: committed progenitor cells. Adapted from: Marciniack-Czorchra A., Stiehl T., Wagner W., Aging 2009 (1) CD34 is a cell surface glycoprotein and functions as a cell-cell adhesion factor. It may also mediate the attachment of stem cells to bone marrow extracellular matrix or directly to stromal cells. In experiments on mice, CD34 has proved useful to distinguish between HSCs with long-term marrow repopulating ability (CD34 low/-ckit + Sca-1 + Lin -), named also (CD34 -KSL), and the progenitors with short-term reconstitution capacity (CD34 + ckit + Sca-1 + Lin -) or (CD34 + KSL). The CD34 -KSL cells express only the following genes GATA-2, IL-1R alpha, IL-2R gamma, AIC-2B (colony stimulating factor 2-receptor, beta, low-affinity [granulocyte-macrophage]), ckit, EPO-R, and c-mpl. In contrast, the CD34 + KSL express all the cytokine receptor genes, which have specificity for haematopoietic lineage, except IL-2R beta, IL-7R alpha, and IL-9R alpha. Clonal culture analysis showed that CD34 -KSL cells were more potent in proliferation and multilineage differentiation capacities than CD34 + KSL cells. Moreover, the clone-sorted CD34 -KSL and CD34 + KSL, cultured in the presence of SCF, IL-3, and EPO showed that CD34 -KSL cells required much more time to undergo the first cell division than CD34 + KSL. Taken together, these data support the claim that CD34 -KSL cells are at a higher rank in haematopoietic hierarchy than CD34 + KSL cells [14]. In humans, CD34 marker could differentiate two populations of HSC: the CD34 -are quiescent cells, which possess colony-forming potential in short-term assays, maintain long-term colony forming potential in in vitro cultures and allow the differentiation of blood cell lineages; and the CD34 + HSC which preserve all these features, but provide the major contribution to haematopoietic engraftment in NOD / SCID mice [9], [10].

(2) Lin -/low signify that HSCs are negative for the markers that are used for detection of lineage commitment [7]. (3) CD38 is a nonlineage-restricted type II transmembrane glycoprotein, which serves as an ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose. The enzymatic functions of CD38 contribute to an array of its immunoregulatory functions. Ligation of CD38 with agonistic antibodies induce diverse effects in haematopoietic cells ranging from growth stimulation to induction and prevention from apoptosis, induction of cytokines production, activation of kinases, and phosphorylation of certain proteins. The CD38 antigen was found to have a rather unique distribution pattern, being predominantly expressed by progenitors, and early haematopoietic cells, then lost during maturation, and re-expressed during cell activation. The expression of membrane CD38 is modulated by certain physiological and pharmacological agents such as cytokines, and lectins [15]. (4) Thy-1 (CD90) expression is restricted to, on average, 1-4% of human foetal liver, cord blood (CB), and bone marrow, and binding to these cells types is essentially restricted to a very small subset of lymphoid cells and approximately 25% of CD34 + cells. The function of Thy-1 on HSCs is unknown, but it has been postulated to be involved in cellular recognition, adherence, and cell activation due to its involvement in the release of intracellular Ca 2+ and phosphorylation of cytoplasmic proteins. Thy-1 may be important in stromal adherence, possibly providing a growth inhibitory signal. The lower expression of Thy-1 on circulating CD34 + cells from umbilical CB may result from a reduced requirement for adherence [16].

I-2.2. HSC Plasticity

Recent evidence has largely hailed the possibility to "transdifferentiate" the plastic bone marrow or circulating stem cells to non-haematopoietic tissue [17]. Transdifferentiation describes the conversion of a cell of one tissue lineage into a cell of an entirely distinct lineage, with concomitant loss of the tissue-specific markers and function of the original cell type, and acquisition of markers and functions of the transdifferentiated cell type [1]. The lineage conversion can occur directly, by activating an otherwise dormant differentiation programme to alter the lineage specificity of the cell (Figure 4A) or could occur via dedifferentiation of a tissue-specific cell to a more primitive, multipotent cell and subsequent redifferentiation along a new lineage pathway (Figure 4B) [1].

The lineage conversion between HSC and neural stem cells (NSC) has been accepted so far. Thus, Sigurjonsson et al. proved that CD34 + HSC from adult human donors could produce neurons efficiently if they are introduced into the lesions of spinal cord of the developing chicken embryo [18]. Moreover, Bjornson et al. showed that genetically labelled NSC, after transplantation into irradiated hosts, could produce a variety of blood cell types, including myeloid and lymphoid cells as well as early haematopoietic cells [19]. The production of differentiated haematopoietic cells is tightly regulated by intrinsic (Figure 5) and extrinsic factors (Figure 6) [20].

Intrinsic regulators

The multipotent haematopoietic cells, prior to differentiation, express genes associated with the erythroid lineage (β globin), and with the myeloid lineage (myeloperoxidase) at low levels, the myeloid transcription factors (PU.1 and C/EBPα), lymphoid (GATA-3), and myeloid receptors such as c-fms, the G-CSF receptor and the alpha chain of the GM-CSF receptor. These observations are consistent with the concept of multilineage priming in stem cells. In this context, lineage commitment requires not only that the appropriate gene expression programme is enhanced, but also that alternate lineages are repressed [20].

Imbalances of this equilibrium induce the disease. For example, the ectopic activation of PU.1 expression in the erythroid lineage inhibits erythroid differentiation and causes erythroleukaemia. The misexpression of the stem cell leukaemia (SCL) transcription factor in T lineage cells conduces to thymocyte differentiation arrest and T-cell leukaemia. The absence of E2A and Pax5 transcription factors disturb the development of committed pro-B cells [20].

To summarise, HSCs express lineage affiliated genes, which are enhanced or repressed upon the degree of maturation [21]. The haematopoietic transcription factors drive cell fate acting cooperatively, as the SCL transcription complex, antagonistic, i.e. the GATA-1 / PU.1, GATA-1 / FOG, or SCL-E2A in the lymphoid lineages, as well as the autoregulatory pathways [20]. The transcriptional regulation is sensitive to intrinsic perturbations.

For example, the E2A transcription factor is essential to B lineage development (commitment and proper B-cell differentiation), and the E2A locus is often concerned by chromosomal translocations in B-cell leukaemias [22]. In the thymus, E2A collaborates with HEB, another basic helix-loop-helix (bHLH) transcription factor, to drive thymocyte differentiation. The oncogenes that are involved in T cell acute lymphoblastic leukaemia (T-ALL) have been shown to directly associate with E2A and HEB in thymocytes, and to inhibit their transcriptional proprieties [23]. Moreover, there are evidences that the B-versus T-cell fate is dependent on the level of E2A activity, B-cell commitment being more sensitive to E2A dosage than T-cell lineage commitment [20].

Other examples include inactivating mutations of GATA-1 in megakaryoblastic leukaemia [24] and of PU.1 in acute myeloid leukaemia (LAM) [25]. In addition, the early lineage commitment is sensitive to PU.1 dosage governing B cell as well as macrophage developments. Thus, low levels of PU.1 are compatible with B-cell fate, while high PU.1 levels is specific to macrophage switch [26]. Moreover, high levels of PU.1 favour the macrophage fate over the granulocyte fate [27].

Extrinsic regulators

Environmental signalling could occur through direct HSC-MSC interaction, or through the action of soluble mediators (Figure 6). In the absence of these signals, haematopoietic cells undergo a default pathway of apoptosis [20]. Thus, there are soluble factors that could contribute to the decision between self-renewal and differentiation. For example, leukaemia inhibitory factor (LIF) through LIF-STAT3 contributes together with bone morphogenetic protein (BMP)-Smad pathway to the decision of maintaining the totipotency of ES in vitro, or, to adopting a neural fate [28]. Other cytokines have a regulator role of haematopoietic cell differentiation through the alteration of transcription factor dosage, thereby shifting the transcription network towards a particular pathway [20]. For example, the dosage of GM-CSF influences the choice between a macrophage and a granulocyte fate, by regulation of PU.1 expression in multipotent CMP, and in committed myeloid progenitors (GMP) [20]. Similarly, the G-CSF receptor induces granulocyte differentiation through upregulation of transcription factor C/EBPα [29] and by altering the ratio of C/EBPα versus PU.1 in favour of the granulocyte lineage [27]. Furthermore, the vascular endothelial growth factor (VEGF) has proved to have an anti-apoptotic role during the development of the primitive erythroid lineage, and this effect is mediated the SCL transcription factor [30].

Similary, c-kit through Jak-STAT signalling pathway suppress apoptosis in haematopoietic cells [31]. Moreover, c-kit prevents progression in the B lineage at the level of transition from pro-B to pre-B by down-regulation of E12 transcription factor, a product of the E2A locus [32]. In conclusion, the combinatorial interaction between cell-extrinsic cues and cell-intrinsic processes consolidates the lineage choice and drive cells to differentiation and maturation pathways. The molecular processes governing hematopoiesis involve the interplay between lineagespecific transcription factors, listed above, and a series of epigenetic tags, including DNA methylation and covalent histone tail modifications [33]. The DNA in eukaryotic chromatin is packed by histones into arrays of repeating units called nucleosomes. Each nucleosome contains a nucleosome core, where the DNA is wrapped around a histone octamer, consists of (H3) 2 -(H4) 2 tetramer and two dimers H2A-H2B, and a stretch of relatively unconstrained DNA called the linker DNA. The nucleosome cores occlude the DNA from many DNA-binding factors, playing a significant role in chromatin packing and gene regulation. In addition, the post-translational modifications of chromatin (such as acetylation, phosphorylation, methylation, ubiquitination, polyADP-ribosylation), commonly known as "histone code", are capable of affecting its structure and gene transcription, and are catalysed by opposing families of enzymes, allowing the developmental potential of HSCs to be dynamically regulated. Thus, during development, in normal conditions, cells undergo a process whereby they lose pluripotency and become committed to a more-restricted cell type. This pathway involves three steps of chromatin remodelling: first, transcriptional repressors are recruited to OCT4; second, heterochromatin is generated by the methylation of H3K9 and the subsequent binding of heterochromatin protein 1 (HP1) to this histone modification; and third, the underlying DNA is methylated [34] (Figure 7). Although the histone modification takes place as a secondary event, the repressive chromatin structure is easily reversed by altering the cell environment, and only the DNA methylation can ultimately stabilizes OCT4, bringing it in the inactive state [34].

Figure 7. Mechanisms involved in disabling the pluripotency genes

In ES cells, pluripotency genes -such as octamer-binding transcription factor 4 (OCT4; named also OCT3 or POU5F1) and NANOG -have unmethylated CpG islands, and the histones that package these genes have specific modifications, including acetylation of histones H3 and H4 and methylation of histone H3 lysine 4 (H3K4). With the onset of differentiation, histone methyltransferase G9A (also known as EHMT2) is recruited, together with a histone deacetylase (HDAC), and this causes deacetylation of local histones and demethylation of H3K4. In the next step, G9A catalyses the methylation of H3K9, and this modification serves as a binding site for the heterochromatin protein 1 (HP1), thus generating a form of local heterochromatin. Finally, G9A recruits DNA methyltransferase 3A (DNMT3A) and DMNT3B, which mediate de novo methylation of the underlying DNA. Ac, acetyl; Me, methyl. From: Cedar H. & Bergman Y., Nature Reviews. Immunology 2011 I-2.4.1. DNA methylation DNA methylation in vertebrates occurs in the cytosine residues of CG dinucleotides, and this process is associated with transcriptional silencing. This silencing can be achieved by either repressing the binding of transcription factors (Figure 8A) or by recruiting proteins that specifically bind to methylated CGs (methyl-CG-binding proteins, e.g., MeCP2), which can further recruit histone deacetyltransferases (HDACs) and corepressors (Figure 8B) [35]. This process is catalysed by a family of enzymes including DNMT1, which preferentially targets hemi-methylated DNA and is required for "maintenance" methylation during DNA replication; and DNMT3A and DNMT3B which are required for de novo methylation [33]. In addition, the 5-methylcytosine dioxygenase TET proteins promote DNA demethylation in mammalian cells through a process that requires the base excision repair pathway [34].

Recent evidence shows that DNA methylation has a direct role in regulating HSC selfrenewal and commitment to lymphoid versus myeloid cell fates [34]. Meis homeobox 1 (Meis1), involved in haematopoiesis lineage development, is unmethylated in multipotent progenitor cells (MPPs), but seems to progressively become hypermethylated and transcriptionally silenced during differentiation progresses [34]. On the other side, many genes in HSCs or MPPs are initially methylated and thereafter undergo to selective demethylation in a lineage-specific manner. For lymphoid lineage, these genes include Lck, which undergoes demethylation in T cells and encodes a SRC family kinase that is responsible for initiating signalling downstream of the T cell receptor, and POU domain class 2-associating factor 1 (Pou2af1), that encodes a B cell-specific co-activator and undergoes demethylation in B cells. In granulocyte-macrophage progenitors, the genes that undergo specifically demethylation are myeloperoxidase (Mpo), and CXC-chemokine receptor 2 (Cxcr2) [34].

I-2.4.2. Histone acetylation and deacetylation

Acetylation is the result of equilibrium between two opposing activities: histone acetyltransferases (HATs) which acetylate the lysine residues on histones and loosen up histone-DNA interactions to allow gene expression, and histone deacetylases (HDACs) that catalyze removal of acetyl groups from lysine residues and strengthen up histone-DNA interactions to prevent gene expression. Acetylation of core histones has been correlated with cellular processes, including chromatin assembly, DNA repair, and recombination (reviewed by Gregoretti I. V.) [36]. The ability of histone acetylation to regulate gene expression occurs via the direct effect of chromatin structure modification, which serves to neutralize the charge between histone tails and the DNA backbone, and also by serving as a docking site for regulatory factors [33]. Moreover, many HDACs are at least partially cytoplasmic; some fractions of these proteins can act on non-histone substrates, including the cytoskeletal protein tubulin and transcription factors (i.e. p53) [36].

During hematopoiesis, lineage-restricted transcription factors regulate specific geneexpression patterns by recruiting HAT or HDAC complexes to the promoters of target genes.

For example, during erythropoiesis, erythroid-specific transcription factors including GATA-1, which is essential for red lineage maturation and survival, directly recruit HAT-containing complexes to the b-globin locus to stimulate transcriptional activation. Specifically, GATA-1 recruits CREB-binding domain (CBP) to the β-globin gene locus, resulting in the acetylation of histones H3 and H4, and facilitating high-globin gene expression. GATA-1 itself is also acetylated on conserved lysine residues by CBP, which leads to enhance its transcriptional activity [33].

I-2.5. Haematopoiesis Functional Assays

Functional tests used to explore haematopoiesis have been conceivable to identify and evaluate separately the various classes of haematopoietic progenitors. Thus, the standardized short-term colony assays quantify the lineage-committed myeloid precursors, but identification of primitive cells, having the ability to repopulate durably myeloid and lymphoid lineages and preserving the self-renew capacity, still largely depends on in vivo assays. Whatever the assay, should measure two cardinal parameters: cell proliferation (measured by the number of cells produced), and the differentiation potential (estimated by the number of different lineages represented in its progeny) [37].

In vitro assays

Short-term in vitro assays

The prototypes of the short-term assays are semi-solid colony assays, or CFC assays, which identify and quantify lineage-restricted progenitors in well-standardized conditions (according to the manufacturer's instructions; see www.stemcell.com/technical/manuals.asp).

The individual progenitors, called colony-forming cells (CFCs), cultured in a suitable semi-solid matrix, such as methylcellulose or collagen supplemented with nutrients and cytokines, proliferate during 14 to16 days at 37°C to form discrete cell clusters or colonies with specific characteristic (composition, size, colour, disposition) (Figure 9). CFU-GEMM: Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte reflect a multipotential progenitor that produces colonies, which contain erythroblasts and cells of at least two other recognizable lineages. Due to their primitive nature, CFU-GEMM tend to produce large colonies of >500 cells (Figure 10 A).

CFU-GM:

Colony-forming unit-granulocyte, macrophage. The colonies contain at least 20 granulocyte cells (CFU-G), macrophages (CFU-M) or cells of both lineages (CFU-GM) (Figure 10 B). CFU-GM colonies arising from primitive progenitors may contain thousands of cells in single or multiple clusters (Figure 10 C).

BFU-E: Burst-forming unit-erythroid. It produces a colony containing >200 erythroblasts in a single cluster or in multiple ones and can be sub-classified based on the number of cells / cell clusters per colony. BFU-E is more immature progenitors than CFU-E and requires EPO and cytokines with burst-promoting activity such as Interleukin-3 (IL-3) and Stem Cell Factor (SCF) for optimal colony growth (Figure 10 D).

CFU-E: Colony-forming unit-erythroid. It produces 1-2 cell clusters containing a total of 8-200 erythroblasts. CFU-Es are mature erythroid progenitors that require erythropoietin (EPO) for differentiation (Figure 10 E).

CFU-Mk:

Colony-forming unit-megakaryocyte (Figure 10 F). These colonies contain 3 or more megakaryocytic cells. Although megakaryocytic progenitors can be cultured in methylcellulose-based medium containing the appropriate growth factors, it is difficult to distinguish CFU-Mk based on cellular and colony morphology (Figure 10 G). Therefore, the manufacturer recommends that CFU-Mk be enumerated in collagen-based, MegaCult®-C following staining of megakaryocytes in dehydrated gels by immunocytochemical staining [38].

Figure 10. Representative pictures for human haematopoietic colonies (Giemsa stain)

Frequencies of CFC among human CD34 + bone marrow cells average 15% in the CD34 + CD38 + fraction, and 5% in the more immature CD34 + CD38 -population, respectively.

[37].

Short-term colony assays are not adequate for the detection of more immature progenitors because the lifespan of these cells in viscous medium (methylcellulose, agar, or plasma clot) does not extend beyond 3 weeks, which is too short a span for stem cells to produce a differentiated progeny, and it cannot be renewed. The semi-solid cultures could be employed to evaluate the myeloid precursors. Surprisingly, the colony assays for lymphoid progenitors have not been well-defined [37].

CFCs can proliferate and generate colonies in liquid cultures, too, in the presence of the same cytokines, but identification of their progeny will require flow cytometry analysis and the benefit of an easy scoring of several progenitors in the same dish would be lost [37].

Long-term assays

In vitro long-term assays for HSCs include Long-Term Culture-Initializing Cell (LTC-IC) assay and Cobblestone Area Forming Cell (CAFC) one. LTC-IC assays are based on the ability of HSCs, but not more mature progenitor cells, to form and maintain a population of progenitor cells with clonogenic potential over at least a five-week culture period [5]. Most human LTC-IC is CD34 + CD38 low/- [37].

CAFC assays measure the ability of the most primitive progenitor cells (in vivo repopulating cells, HSCs, and day 12 CFU-S) to grow under stromal cell layer where they proliferate cyclically and release their progeny (CFC and differentiated cells) in the nonadherent fraction (Figure 11) [5], [37]. These cells could be maintained for several months in mice and 8 to 10 weeks in humans [37]. The bone marrow microenvironment is an ensemble of structures, which provide the basic framework, nutritional supply, the waste removal system for haematopoiesis, and the specialized support of self-renewal and differentiation for the haematopoietic precursors.

This system belongs to the vascular network, bone marrow innervation, bone marrow (BM) stroma, and extracellular matrix.

The marrow vasculature consists principally of a network of sinuses that originate at the endosteum from cortical capillaries and terminate in collecting vessels that enter the systemic venous circulation [39]. The marrow does not have lymphatic drainage [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF].

Bone marrow innervation occurs with myelinated and non-myelinated nerves that enter through the nutrient canals. Some innervation also occurs through epiphyseal and metaphyseal foramina. Nerve bundles follow the arterioles with branches serving the smooth muscle of the vessels or, occasionally, terminating in the haematopoietic tissue amongst haematopoietic cells. [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF].

I-3.2. Histology of the Bone Marrow

The bone marrow in adults is found within the cancellous bone of the axial skeleton, girdle bones, and some areas of the metaphyses [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF].

It consists of haematopoietic tissue islands surrounded by vascular sinuses interspersed within a meshwork of trabecular bone.

Trabecular bone is enclosed by a complex layer of diverse cells:

-osteoblasts, -osteoclasts, -and flat, simple or two-layered, nonactivated bone-lining cells (BLCs) [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF].

The trabecular surfaces are covered by a layer of endosteal cells. Occasional multinucleate osteoclasts are also a normal finding at trabecular margins [START_REF] Wilkins | Histology ofnormal haemopoiesis: Bone marrow histology I[END_REF].

The inner surface of the bone cavities and the outer surface of the cancellous bone spicules within the cavities are covered by an endosteal lining consisting of a single layer of flat "bone-lining cells" supported by a thin layer of reticular connective tissue, osteoblasts and osteoclasts (Figure 12) [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF]. The sinuses (vascular spaces) of the marrow are thin-walled, lined with flat specialized endothelial cells, which prevent the premature escape of immature cells into the peripheral blood. The basal lamina of venous sinuses is incomplete and in combination with a thin or not basement membrane, allowing mature cells to pass through the wall of the sinuses [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF], [START_REF] William | PDQ Series[END_REF].

Haematopoietic tissue islands (Haematon Units / Buffy coat) are composed from haematopoietic cells in interaction with the bone marrow microenvironment to form the socalled bone marrow haematopoietic stem cell niches.

Haematopoietic stem cells and lineage-restricted progenitor cells (HPCs) are not randomly distributed in the BM, but there are localized according to their differentiation stage. The majority of true HSCs are found at the endosteum in contact with osteoblasts, whereas more committed progenitors accumulate in the central BM [START_REF] Lévesque | Bone Marrow-Derived Progenitors[END_REF]; erythropoiesis takes place in distinct anatomical units (erythroblastic islands), granulopoiesis occurs in less distinct foci, and megakaryopoiesis occurs adjacent to the sinus endothelium [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF]. The barrier cells hold many haematopoietic cells, notably putative stem cells and differentiating megakaryocytes.

The massed barrier cells are disposed in a crescent, progressing deep into the marrow. Barrier cells, especially in haematopoietic zones containing very early differentiating stages, may insinuate long, slender processes between and around endothelium and adventitial tunics. The blood-marrow barrier is thereby augmented, impeding emigration and immigration of circulating cells, preventing the premature release of immature haematopoietic cells to the circulation as well. In contrast, profiles of vascular sinuses can be made entirely of a simple layer of barrier cells stretched quite thin, except at the perikaryon. These lie in haematopoietic zones containing late differentiating forms ready for delivery to the circulation, their wall being crossed by blood cell-filled apertures. They are structurally suited to facilitate delivery of blood cells to the circulation [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF].

Stromal Cells includes all cell types that are located between the outer surfaces of marrow blood vessels and the bone surfaces that encase the haematopoietic space and tissue [START_REF] Krebsbach | Bone marrow stromal cells: characterization and clinical application. Critical reviews in oral biology and medicine[END_REF]. The stromal cells compose the supportive tissues of the bone marrow [START_REF] Wilkins | Histology ofnormal haemopoiesis: Bone marrow histology I[END_REF].

In this family are included:

• Westen-Bainton cells / adventitial reticular cells / stromal fibroblasts / barrier cells / pericytes (modified fibroblasts that produce the reticulin framework of the bone marrow)

• adipocytes ( cells which store energy in the form of fat)

• endothelial cells

• bone lining cells (inactive osteoblasts [45] ), osteocytes [41] , and osteoblasts [40] .

Westen-Bainton cells, adventitial reticular cells (ARCs), pericytes, stromal fibroblasts, and barrier cells cover two sides on the surface of bone, in particular the surface of bone to the adventitial surface of vascular sinuses and extend en bloc into the marrow [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF]. Their name as well as their origin raises vivid controversy. Thus, after Krebsbach et al. the Westen-Bainton cells (name attributed by Westen and Baiton in 1979) are referred to by a variety of descriptive terms (reticular cells, adventitial reticular cells, and stromal fibroblasts (Figure 13) [START_REF] Krebsbach | Bone marrow stromal cells: characterization and clinical application. Critical reviews in oral biology and medicine[END_REF]. The recent evidence claimed that MSCs, named also CFU-F, are either identical to or derived from pericytes, if it taking into account the similarities in their physical relationship to the vasculature, the cellular response to growth factors, and expression of similar phenotypic markers [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF], [START_REF] Crisan | A perivascular origin for mesenchymal stem cells in multiple human organs[END_REF], [START_REF] Sacchetti | Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment[END_REF], [START_REF] Da | In Search of the In Vivo Identity of Mesenchymal Stem Cells[END_REF]. Moreover, Bianco P et al. suggested in 2001 that ARCs themselves "can be seen as bona fide specialized pericytes of venous sinusoids in the marrow" linking both cells together [START_REF] Bianco | Bone marrow stromal stem cells: Nature, biology, and potential applications[END_REF]. On the other hand, another controversial hypothesis has recently held that pericytes may arise directly from endothelial cells or their progenitors [START_REF] Deruiter | Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro[END_REF], [START_REF] Yamashita | Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors[END_REF].

In terms of morphology, there are similarities between BM MSCs with long projection and BM adventitial reticular cells (Figure 13) [START_REF] Jones | Human bone marrow mesenchymal stem cells in vivo[END_REF]. Another suggestive description, cells with "extensive, elongated, and attenuated cell processes" support the alternative use of the term "reticular" [START_REF] William | PDQ Series[END_REF], [START_REF] Krebsbach | Bone marrow stromal cells: characterization and clinical application. Critical reviews in oral biology and medicine[END_REF].

Likewise, in terms of phenotype, both types of cells are similar, the MSCs sorted based on STRO-1 + CD106 + or STRO-1 + CD146 + phenotype expressed α smooth muscle actin or were positive for 3G5 antigen, which are considered to be specific for pericytes [START_REF] Jones | Human bone marrow mesenchymal stem cells in vivo[END_REF], [START_REF] Gronthos | Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[END_REF], [START_REF] Shi | Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp[END_REF].

Other phenotypic markers shared by ARCs, BLCs, and MSCs STRO-1 + CD106 + CD146 + are: CD271 (low-affinity nerve growth factor receptor [LNGFR]), stromal cell-derived factor-1 (SDF-1) or Chemokine (C-X-C motif) ligand 12 (CXCL12), and several teams include also, few stromal / fibroblast markers: CD10, CD13, D7-FIB [START_REF] Jones | Human bone marrow mesenchymal stem cells in vivo[END_REF], [START_REF] Gronthos | Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[END_REF]. Moreover, cytochemical stain proves their positively for alkaline phosphatase [START_REF] Jones | Human bone marrow mesenchymal stem cells in vivo[END_REF], [START_REF] Gronthos | Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[END_REF], types I and III collagen, and osteonectin [START_REF] Krebsbach | Bone marrow stromal cells: characterization and clinical application. Critical reviews in oral biology and medicine[END_REF].

Another observation was that pericytes isolated from bovine retinal capillaries are STRO-1 + and exhibit the potential for differentiation into a variety of cell types including osteoblasts, adipocytes, chondrocytes, and "more mundane fibroblasts" [START_REF] Short | Mesenchymal Stem Cells -Review Article[END_REF], [START_REF] Schor | Pericyte differentiation[END_REF], [START_REF] Doherty | Vascular pericytes express osteogenic potential in vitro and in vivo[END_REF]. Presumptive pericytes in every developing tissue also express angiopoietin-1, which signals through Tie-2 expressed by endothelial cells, and this signalization seems to be indispensable for vessel stabilization and integrity and for pericyte recruitment [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF], [START_REF] Suri | Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis[END_REF]. The barrier cells are activated cells, displaying organelles associated with intense protein synthesis and secretory activity [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF].

Recently, evidence has been produced that phenotype-purified human ARCs are able to generate osteoblasts [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF] and adipocytes [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF], [START_REF] Serghei | The interplay of osteogenesis and hematopoiesis: expressin of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow[END_REF] and to self-renew into new adventitial reticular cells and CFU-Fs in vivo [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF].

Apparently, both in humans and mice, perisinusoidal "reticular" cells (human ARCs, and murine CXCL 12-abundant reticular (CAR) Nestin + cells, which seem to be equivalent populations) play a significant role in regulating HSCs, also [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF], [START_REF] Méndez-Ferrer | Mesenchymal and haematopoietic stem cellsform a unique bone marrow niche[END_REF].

Reticular cells send branches stretching from the bone surface to the adventitial surface of vascular sinuses (Figure 13, sinus 2) [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF]. These cells synthesize reticular (argentophilic) fibres that, along with their cytoplasmic processes, extend into the haematopoietic compartments and form a meshwork on which haematopoietic cells rest. The cell bodies, their broad processes, and their fibres constitute the reticulum of the marrow [39].

Adipocytes

In postnatal marrow, the marrow adipocytes (Figure 15) develop directly from ARCs around sinusoids (Figure 14) [START_REF] Bianco | Bone and the hematopoietic niche: a tale of two stem cells[END_REF].

From: Bianco P., Blood 2011

Adipocytes express leptin, osteocalcin and prolactin receptors during their differentiation, thereby promoting haematopoiesis and influencing osteogenesis [39], [START_REF] Laharrague | High expression of leptin by human bone marrow adipocytes in primary cultures[END_REF], [START_REF] Benayahu | Osteocalcin (BGP), gene expression, and protein production by marrow stromal adipocytes[END_REF], [START_REF] Mcaveny | Prolactin receptor expression during adipocyte differentiation of bone marrow stroma[END_REF], [START_REF] Gimble | The function of adipocytes in the bone marrow stroma: an update[END_REF].

Adipocyte maturation in vitro is inhibited by stromal-derived cytokines such as IL-1 and IL-11 [39], [START_REF] Delikat | IL-1 beta inhibits adipocyte formation in human long-term bone marrow culture[END_REF], [START_REF] Keller | Interleukin-11 inhibits adipogenesis and stimulates myelopoiesis in human long-term marrow cultures[END_REF].

Marrow adipocyte leptin may modulate adjacent haematopoietic progenitor growth [39], [START_REF] Tavassoli | Fatty evolution of marrow and the role of adipose tissue in hematopoiesis. Handbook of the Hemopoietic Microenvironment[END_REF].

Adipocyte differentiation by marrow stromal cells is inhibited by bone morphogenetic proteins and leptin, supporting the reciprocal regulation of osteogenesis and adipogenesis in the marrow microenvironment [39], [START_REF] Gimble | Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells[END_REF], [START_REF] Thomas | Leptin acts on human marrow stromal cells to enhance differentiation to osteoblasts and to inhibit differentiation to adipocytes[END_REF].

Endothelial cells

Endothelial cells (Figure 15) of capillary vessels are responsible for regulating of HSCs traffic, allowing to haematopoietic mature cells to pass in circulation. Through the synthesis of growth factors, they also contribute to boosting haematopoiesis. Likewise, they enhance bone regeneration in the osseous defects; by their involvement in the formation of blood vessels that supply oxygen and nutrients to developing bone tissue. However, it has been suggested, more recently, that endothelial cells may play a more direct role in bone development and formation, through their interactions with osteoprogenitor cells and, under certain conditions, their production of specific bone inductive factors [START_REF] Lévesque | Bone Marrow-Derived Progenitors[END_REF], [START_REF] Kaigler | Transplanted Endothelial Cells Enhance Orthotopic Bone Regeneration[END_REF]. Marrow endothelial cells express von Willebrand factor antigen [39], [START_REF] Hasthorpe | Characterization of endothelial cells in murine long-term marrow culture: Implication for hemopoietic regulation[END_REF], type IV collagen, and laminin [39], [START_REF] Perkins | Stromal cell progeny of murine bone marrow fibroblast colony-forming units are clonal endothelial-like cells that express collagen IV and laminin[END_REF]; they also constitutively express two adhesion molecules: VCAM-1 and Eselectin [39], [START_REF] Schweitzer | Constitutive expression of Eselectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues[END_REF]. Other receptors that may mediate marrow cellular trafficking include fractalkine, an endothelial membrane-bound chemokine, also upregulated by cytokines [39], [START_REF] Imai | Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion[END_REF]. Marrow sinusoidal endothelium specifically expresses sialylated CD22 ligands, which are homing receptors that recirculate B lymphocytes [39], [START_REF] Nitschke | Identification of CD22 ligands on bone marrow sinusoidal endothelium implicated in CD22-dependent homing of recirculating B cells[END_REF].

Bone cells

Osteoblasts, osteoclasts, and elongated flat cells with a spindle-shaped nucleus (flat bonelining cells) form the marrow endosteal lining [39], [START_REF] Deldar | Bone lining cells and hematopoiesis: an electron microscopic study of canine bone marrow[END_REF]. Resting endosteal cells express vimentin, tenascin, alpha smooth-muscle actin, osteocalcin, CD51, and CD56 [39], [START_REF] Sillaber | Immunophenotypic characterization of human bone marrow endosteal cells[END_REF].

Cultured human bone cells express high levels of α1/β1, α3/β1, and α5/β1 integrins [39], [START_REF] Saito | Identification of integrin receptors on cultured human bone cells[END_REF].

Endosteal cells provide a homing niche for newly transplanted haematopoietic stem cells [39], [START_REF] Nilsson | Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model[END_REF]. Mesenchymal stromal cells positive for the STRO-1 antibody can differentiate into adipocyte, chondrocytic, and osteogenic cells [39], [START_REF] Park | Interconversion potential of cloned human marrow adipocytes in vitro[END_REF], [START_REF] Dennis | A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse[END_REF], [START_REF] Mark | Multilineage Potential of Adult Human Mesenchymal Stem Cells[END_REF], [START_REF] Oyajobi | Isolation and characterization of human clonogenic osteoblast progenitors immunoselected from fetal bone marrow stroma using STRO-1 monoclonal antibody[END_REF], and similar osteogenic potential is found in STRO-1 positive vascular pericytes [39], [START_REF] Doherty | Vascular pericytes express osteogenic potential in vitro and in vivo[END_REF]. This process of mesenchymal stromal cell to osteogenic differentiation is associated with the loss of the activated leukocyte adhesion molecule (CD166) [39], [START_REF] Bruder | Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation[END_REF].

Osteoblasts

Bone-forming osteoblast progenitor cells (Figure 15), like stromal precursors, reside in the CD34 negative, STRO-1 positive marrow cell population [39], [START_REF] Long | Regulation of human bone marrowderived osteoprogenitor cells by osteogenic growth factors[END_REF], [START_REF] Gronthos | Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells[END_REF], [START_REF] Stewart | Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells[END_REF]. Bone morphogenetic protein 2, b-FGF, and TGF-b promote the growth and differentiation of these cells [39], [START_REF] Long | Regulation of human bone marrowderived osteoprogenitor cells by osteogenic growth factors[END_REF], [START_REF] Hanada | Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells[END_REF].

Osteoblasts expand early haematopoietic progenitor survival in long-term cultures and secrete haematopoietic growth factors such as macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), GM-CSF, IL-1, and IL-6 [39], [START_REF] Taichman | The role of osteoblasts in the hematopoietic microenvironment[END_REF], [START_REF] Ahmed | Cytokine-induced expansion of human CD34 + stem/progenitor and CD34 + CD41 + early megakaryocytic marrow cells cultured on normal osteoblasts[END_REF].

Osteoblasts also produce haematopoietic cell-cycle inhibitory factors such as TGF-b, which may contribute to their intimate role in stem cell regulation within the marrow microenvironment [39], [START_REF] Robey Robey | Osteoblasts synthesize and respond to transforming growth factor-type b (TGF-beta) in vitro[END_REF].

Osteoclasts

Bone-resorbing osteoclasts (Figure 15) are derived from haematopoietic progenitors (CD34 positive, STRO-1 negative) and branches from the monocyte-macrophage lineage early during the differentiation [39], [START_REF] Matayoshi | Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells[END_REF], [START_REF] Roodman | Cell biology of the osteoclast[END_REF]. KIT ligand and M-CSF act synergistically on osteoclast maturation [39], [START_REF] Demulder | Effects of stem cell factor on osteoclast-like cell formation in long-term human marrow cultures[END_REF], and M-CSF is essential for the proliferation and maturation of osteoclast progenitors [39], [START_REF] Tanaka | Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors[END_REF]. Osteoprotegerin (OPG), or osteoclastogenesis inhibitory factor, is a cytokine of the tumour necrosis factor receptor superfamily, which inhibits osteoclast differentiation [39], [START_REF] Shalhoub | Osteoprotegrin and osteoprotegrin ligand effects on osteoclast formation from human peripheral blood mononuclear cell precursors[END_REF]. Osteoclast maturation requires osteoprotegerin ligand (TRANCE / RANKL), an osteoclast differentiation and activation factor (ODF) elaborated by stromal cells and osteoblasts [39], [START_REF] Takahashi | A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function[END_REF]. Cross-linking antibodies to the adhesion receptor CD44 inhibit osteoclast formation in primary marrow cultures treated with 1 alpha 25dihydroxyvitamin D3 [39], [START_REF] Kania | CD44 antibodies inhibit osteoclast formation[END_REF]. Similarly, blocking the expression of cadherin-6 interferes with heterotypic interactions between osteoclasts and stromal cells, impairing their ability to support osteoclast formation [39], [START_REF] Mbalaviele | Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation[END_REF].

Accessory cells (monocytes, macrophages, T cells) are progeny of haematopoietic stem cells [100]. Macrophages fulfil functional roles: haematopoietic growth factors synthesis, store iron for haemoglobin production, and carry out phagocytosis of debris [START_REF] Travlos | Normal Structure, Function, and Histology of the Bone Marrow[END_REF]. Extracellular matrix (ECM) molecules are synthesized and secreted by microenvironmental cells and include collagens (types I, III, IV, and V), glycoproteins (fibronectin, laminin, thrombospondin, hemonectin, and tenascin), and glycosaminoglycans (hyaluronic acid and chondroitin, dermatan, and heparan sulfate). Besides providing structure to the marrow space, and a surface for cell adhesion, the microenvironment is important for haematopoietic cell homing, engraftment, migration, and the response to physiologic stress and homeostasis

I-3.3. Mesenchymal Stromal Cells

Mesenchymal stromal cells are progenitors of skeletal tissue components, such as bone, cartilage, the haematopoiesis-supporting stroma, and adipocytes. In addition, they could be experimentally induced to undergo neural or myogenic differentiation [START_REF] Bianco | Bone marrow stromal stem cells: Nature, biology, and potential applications[END_REF]. Therefore, the literature surrounding the MSC is challenging and valuable information could be lost (Table 1).

I-3.

I-3.3.2. Cytomorphology and cytomorphometry

The primary low density plated cultures from BM MSCs showed heterogeneous groups of large flat cells, smaller spindle-shaped cells, and small round cells (Figure 16) [107], [108],

[109], [110]. A size-sieved method, using a 3-µm pore plate, also allowed the isolation of two populations.

The first was a population of cells with a size greater than 3 µm. Cells in this population revealed a fibroblast-like morphology, self-renewal capacity, and multilineage potential, CD73 it has been demonstrated to be highly specific to characterize MSCs, and useful for the development of robust in vitro MSC assays [START_REF] Jones | Human bone marrow mesenchymal stem cells in vivo[END_REF], [START_REF] Mark | Multilineage Potential of Adult Human Mesenchymal Stem Cells[END_REF]. However, another marker, STRO-1, is worth noting, because it proved to have an enrichment capacity in CFU-Fs approximately 100-fold when compared with MSCs isolated by plastic adherence [START_REF] Gronthos | Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[END_REF], [123].

Nonetheless, even though MSCs are widely thought to be CD45 negative, note that other There are eight genes whose expression is increased during all three mesenchymal lineage differentiations (multi-, bi-, and unipotent), suggesting that they represent the putative master control genes. These genes were identified as period homolog 1 (PER1), nebulette (NEBL), Their commitment to a lineage can be induced by a number of stimuli (Table 3) [133]. It is difficult to establish a hierarchy between MSC subpopulation, and apparently, there are differences between the proper MSCs and the plastic adherent cells that are isolated from marrow using different protocols and assigned different names. To date, the marrow-isolated adult multilineage inducible (MIAMI) cells that were isolated from marrow of vertebral Of the first category, the broad investigated markers were STRO-1, and CD73.

Although stromal precursor antigen-1 (STRO-1) is widely regarded as a marker of early mesenchymal stromal precursor cells, it is also expressed on the surface of other human bone marrow (BM) cells that include glycophorin-A + nucleated red cells and a small subset of CD19 + B-cells; however, it is not expressed on HSCs [123]. This has raised many questions about its use as a specific marker in MSC sorting protocols [START_REF] Jones | Human bone marrow mesenchymal stem cells in vivo[END_REF] expression in both tumour and host cells protects the tumour from incoming anti-tumour T cells and suppresses T cell functions through the CD39 (ecto-ATPase)-CD73 axis [19]. Much less is known about CD73 role in MSC biology, but its impact on cell-matrix interactions in chicken fibroblasts has been described [152].

The gold standard assay utilized to identify MSCs is the colony forming unit-fibroblast (CFU-F) assay that, at minimum, identifies adherent, spindle-shaped cells that proliferate to form colonies [153].

In this respect, in the following lines, we present the protocol provided by StemCell Technologies.

Processing of Cells and the CFU-F Assay

1. When working with a fresh bone marrow (BM) sample, the cells need to be processed to remove the red blood cells or enrich desired cells prior to culture. Choose one of the following methods:

• Ammonium chloride lysis (to remove the red blood cells)

• Isolation of the mononuclear cells by Ficoll-Paque™ density gradient separation

• Enrichment of mesenchymal stem cells using the RosetteSep® Human Mesenchymal Stem Cell Enrichment Kit 2. After processing, wash the cells by adding 10 mL of PBS with 2% FBS to the cell pellet.

Centrifuge the cells at 300 x g (~1200 rpm) for 10 minutes at 20°C. Remove the supernatant and resuspend the cells in 1 -2 mL of Complete MesenCult® Medium (Human).

3. If working with BM cells processed with ammonium chloride or Ficoll-Paque™, perform a nucleated cell count (using 3% Acetic Acid with Methylene Blue) and dilute the cells to a stock cell concentration of 2 x 106 cells/mL in Complete MesenCult® Medium (Human).

If working with enriched mesenchymal cells isolated using the RosetteSep® Enrichment Kit for Human Mesenchymal Stem Cells, perform a cell count and dilute cells to a stock cell concentration of 5 x 105 cells/mL in Complete MesenCult® Medium (Human).

4. Plate three different cell densities by adding 1.0 mL, 0.5 mL, and 0.25 mL of the cells at stock concentration to separate 100 mm tissue culture-treated dishes (or T-25 cm 2 tissue culture flasks) prefilled with Complete MesenCult® Medium (Human) to a total volume of 10 mL. For ficolled or lysed BM cells, this will yield final cell count of 2 x 10 6 cells, 1 x 10 6 cells and 0.5 x 10 6 cells in 10 mL of medium. For RosetteSep®-enriched cells this will yield final cell count of 5 x 10 5 cells, 2.5 x 10 5 cells and 1.25 x 10 5 cells in 10 mL of medium.

Plating three concentrations will ensure that the resulting numbers of colonies can be scored, as there are differences in the proliferative potential of CFU-F from various bone marrow samples.

5. Place the 100 mm dishes (or T-25 cm 2 tissue culture flasks) into a 37°C humidified incubator with 5% CO2 in air and >95% humidity for 14 days.

Maximum colony size and numbers are typically observed at 14 days [154].

Suggested Procedure for Staining and Enumeration of CFU-F-Derived Colonies

Staining 1. Remove the medium from cultures of CFU-F grown in 100 mm tissue culture dishes or T-25 cm 2 tissue culture flasks and discard into the bio-hazardous waste. The adherent colonies will remain attached to the plate.

2. Wash the culture dishes or flasks twice using PBS to remove any remaining medium.

Discard the PBS from the two washes into the bio-hazardous waste.

PE is the ratio of the number of colonies to the number of cells seeded:

Proliferative potential → Proliferation assay Doubling time (time for one mitosis / division) → t/n, t = time for the MSCs plated at 1000 cells / 25 cm 2 to reach 80% of confluency, n = number of population doublings (means number of mitosis necessary to reach at 80% of confluency). The number of population doubling is calculated using formula n = log (x/y)/ log 2, x = number of cells plated initial, y = number of cells at 80% of confluency.

Multilineage differentiation potential of MSCs is usually evaluated by the MSCs capacity to differentiate into three main lineages: osteogenic, adipogenic, and chondrogenic (Figure 19). The haematopoietic niche comprises complex interactions between multiple cells and molecules.

A key player of these interactions is the osteoblast. This was proved by Nilsson S. K. et al., using labelled HSCs that had been transplanted in vivo, and were re-found near the endosteum, where osteoblasts are also located [155]. In addition, the osteoblasts ablation results in a rapid and strong reduction in BM cellularity and a ten-fold reduction in the number of HSCs present in the BM [START_REF] Lévesque | Bone Marrow-Derived Progenitors[END_REF].

Lodgement at the endosteal niche is driven by a calcium gradient and an array of osteoblastmediated adhesive interactions [START_REF] Lévesque | Bone Marrow-Derived Progenitors[END_REF]. The osteoblasts promotes HSCs migration to and lodgement at the endosteal HSC niche through adhesive molecules which it produces such as osteopontin, N-cadherin, transmembrane c-KIT ligand stem cell factor (tm-SCF), and the polysaccharide hyaluronic acid involved in HSCs keeping at the endosteum [START_REF] Lévesque | Bone Marrow-Derived Progenitors[END_REF].

Moreover, they signalize and regulate the survival / quiescence / proliferation of HSCs through expression of some receptors. Thus, through parathyroid-related peptide receptor (PTH1R) and activation of the protein kinase A (PKA) pathway, increase the Jagged 1 Recently, an "endothelial HSC niche" has been described [START_REF] Lévesque | Bone Marrow-Derived Progenitors[END_REF]. Thus, some HSCs that express CD150 are found in direct contact with the many endothelial sinuses that irrigate the BM. It is not clear, however, whether these HSCs that locate to endothelial sinuses represent cells in transit (as HSCs permanently leak into the circulation and home back to the BM) or whether they represent a separate HSC pool that is regulated differently. What is clear, however, is that these HSCs that lodge in specific endothelial niches must utilize a separate array of adhesive interactions from the HSCs. Indeed, unlike osteoblasts, BM endothelial cells that compose this endothelial niche express neither osteopontin, nor N-cadherin. Instead, they express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), VCAM-1/CD106, P-selectin, and E-selectin whose ligands are all expressed at the surface of HSCs. Similar to the adhesion molecules expressed in the endosteal niches, P-and E-selectinmediated adhesion regulates HSCs survival, proliferation, and differentiation. Thus, the two anatomically distinct HSC niches, endosteal and endothelial, must regulate HSC turnover differently [START_REF] Lévesque | Bone Marrow-Derived Progenitors[END_REF].

I-4. Conclusion

Recent data implicate cytokines, growth factors, adhesion molecules and signalling receptors as key elements in detecting and translating the extrinsic cues provided by the haematopoietic microenvironmental niches. Thus, these microenvironmental factors supply a specific set of molecules that determine or regulate the haematopoietic stem cell fate (Figure 21). Chapter II-The Myelodysplastic Syndromes

II-2. Clonal hematopoiesis in Myelodysplatic Syndromes

To date, the dilemma of clonality seems far from being resolved. A clonal nature of the myeloid lineage is largely accepted, but the involvement of a lymphocytic lineage remains debatable [170]. The MDS clonality seems to be the result from a pre-malignant or malignant transformation, unlike the benign nature of haematopoiesis clonality from aplastic anaemia, and from paroxysmal nocturnal haemoglobinuria, where it is assumed that is due to

From: Tiu R. et al., Leukemia 2007

If the clonal defect arises in a stem cell, all three lineages derived from that stem cell carry the lesion. Most of the lesions do not involve the lymph-haematopoietic stem cells, thus the lymphocytes are usually polyclonal. In some cases, the progenitor cells rather than stem cells are the initial source of clonal evolution. Both stem cell and progenitor-derived clones can variably contribute to the formation of mature progeny production.

However, this hypothesis (mechanical displacement of normal haematopoiesis) cannot explain the mechanism of cytopenia installed early in the disease.

Of note is, while telomeric erosion may accompany the persistence of a singular clone, by itself, telomere shortening is not a limiting factor for the long-term function of the defective stem cell. Likewise, activation of telomerase activity may be a part of this process, preventing simply depletion of the malignant stem cell [171].

In MDS, clonal evolution has a malignant character and is associated with the stepwise acquisition of a more aggressive phenotype, likely due to secondary defects acquired in the However, in MDS the aberrant clone is capable to produce mature progeny. Thus, the differentiated progeny may correspond to a mixture of both clonal and normal cells, and the ability to produce differentiated progeny may depend on the type of the genetic lesion that in certain circumstances may result in a total maturation block. Of note is that lack of a clonal marker in all or some of the mature cells may not exclude clonality. Instead, a pathogenic chromosomal abnormality may just be a secondary lesion affecting only a subclonal population [171].

Another possibility is that the primary lesion is acquired at the level of the haematopoietic stem cells, while additional, secondary defects occur in progenitor cells [171].

II-3. Aetiology and pathophysiology of Myelodysplatic

Marrow Microenvironment

To date, the role of microenvironment in MDS pathogenesis is controversial, being largely considered a disease of HSC. However, in recent years there is increasing evidence about the cellular microenvironment involvement in the pathogenic process, both by the secretory component and especially by direct interaction with HSC. Thus, in a previous study, Boudard et al. have reported growth defects of stromal cells isolated from MDS patients, resulting in low confluency, associated in some cases with caspase-3 activation and increases of angiogenesis [190]. The present work was focused on cell adhesion-mediated MDS pathogenesis.

Apoptosis, Dysplasia

Despite increased proliferation of the BM cells, there is an increased rate of programmed cell death. Indeed, some of the dysplastic appearance may be explained by apoptotic changes

[175], [191], [192].

Certain types of behaviour related to apoptosis within the MDS disorders can be distinguished:

-in refractory anaemia (RA) / refractory anaemia with ringed sideroblasts (RARS), apoptosis always exceeded proliferation, whereas in refractory anaemia with excess of blasts (RAEB), this ratio equalized on account to increased proliferation;

-progression to RAEB in transformation (RAEB-t) / MDS-related acute myeloid leukaemia (AML), or MDS/AML was associated with a significant reduction in apoptosis, and proliferation;

These observations are supported by the fact that in RA / RARS compared with normal settings, the ratio between pro-apoptotic (Bax/Bad) versus anti-apoptotic (Bcl-2/Bcl-X)

proteins is increased, whereas the disease progression was associated with a significant decrease of this ratio, due primarily to Bcl-2 high expression [193], [194].

Likewise, Kurotaki H and colleagues noticed that the suppression of apoptosis is the result of enhances bcl-2 expression besides the p53 accumulation in haematopoietic precursors selected from MDS-related acute myeloid leukaemia [195].

Moreover, an increased expression of CD95 (Fas) and CD95L (Fas-L) was noticed in CD34 + cells selected from the newly diagnosed MDS patients [196], [197], [198].

The increased apoptosis observed in MDS was correlated also with increased levels of caspase-3, which are significantly higher in RA and RARS than in chronic myelomonocytic leukaemia (CMML), RAEB and RAEB-t [199], [200]. There are evidences that inhibitors of this pathway block tumour homing and engraftment, and reverse the cell adhesion-mediated drug resistance of tumours.

Cytokines

Other evidence is that integrin expression pattern is altered in tumour cells. They are involved in pathogenic process in two ways, i.e., by increasing the proliferation, migration and survival of cancerous cells, and by adhesion-mediated quiescence, which may contribute to the failure of standard cytotoxics in eradication of the tumour cells adhered to bone marrow stromal cells or to the extracellular matrix. Several FISH strategies could increase the method sensibility, such as combined control and target probes to detect genomic deletions or amplifications, dual fusion probes to identify the specific translocations, and break-apart probes to evaluate gene rearrangements [229].

However, the FISH is more reliable for the detection of duplications rather than deletions of Contribute to clonal dominance [233] Decreased survival [233] TET2 4q NA Impaired 5hmC production * [233] Unknown [233] IDH1/IDH2 2q/15q NA Synthesis of 2hydroxyglutarate which impares histone demethylase activity; Inhibit 5hmC production by TET2 [233] Decreased survival [233] Mutations affecting histone function EZH2 ASXL1 20q NA Regulation of histone function [233] Poor prognosis [236]

Secondary MDS and MDS/AML or Therapy-related MDS

TP53

17p NA Mutations in the tumour suppressor [234] Decreased survival [233]

RUNX1

21q NA Impairment of HSC differentiation [234] Decreased survival [233] N/KRAS 1p/12p NA Mutations in the signal transducer RAS [234] Unknow [233] FLT3-ITD 13q CD34 + cells Impairment of HSC differentiation [234] Decreased survival [233] NPMc 5q NA Impairment of normal cellular homeostasis [234] Unknow [233] MDS/MPN Overlap Syndromes CBL 11q NA Marker of myelomonocytic clonal dominance without impact on clinical outcome [236] Unknow [233] JAK2 9p NA Myeloid neoplasm associated with ringed sideroblasts and/or thrombocytosis [235] Unknow [233] * 5'-hydroxymethylcytosine (5hmC); DNA methyltransferase converts unmethylated cytosine to 5mC. 5mC is converte to 5hmC by the TET proteins in the presence of αKG generated by the IDH enzymes; encodes for a protein SF3B1, which associates with SF3A and 12S RNA to form the U2 small nuclear ribonucleoprotein particles, and that is believed to play a role in pre-mRNA splicing and associated transcription [237]. The occurrence of a somatic mutation of SF3B1 causes mitochondrial iron overload, ineffective erythropoiesis, and anemia, typical myelodysplastic features of RARS. The subsequent occurrence of a somatic mutation of JAK2 or MPL involves the acquisition of myeloproliferative features, including thrombocytosis (Figure 24)

[235]. A potential explanation for this disadvantage is the selection of an "unstable" MSC clone which may facilitate the expansion of malignant cells [230].

II-5. Classification

The MDS classifications were conceived to perform a prognostic stratification of these heterogeneous entities. The initial FAB (French-American-British) system developed in the mid-1970s proposed the MDS subgroups definition using the cellular morphology. The FAB system focused on three criteria: percentages of blasts in BM, PB, and of BM sideroblasts. -grouping in the "Refractory cytopenia with unilineage dysplasia (RCUD)"category of all cases which exhibit unilineage myeloid dysplasia;

-removing the "RCMD-RS = refractory dysplasia with multilineage dysplasia and ringed sideroblasts" entity;

-substituting "CMML = Chronic myelomonocytic leukaemia" with Myelodysplastic / myeloproliferative neoplasm (MDS / MPN);

-employing the percentages of circulating blasts to discriminate RAEB-1 of MDS unclassified (MDS-U);

-introducing the entity "Therapy-related secondary myeloid neoplasms" to regroup the AML and MDS chemotherapy related cases; -adding a provisional entity, refractory cytopenia of childhood (RCC), which include children with cytopenia(s) with less than 2% blasts in the peripheral blood and less than 5% in the bone marrow and evidence of dysplasia in two or more lineages (

II-6. Prognostic

Throughout the years, a decisive observation, such as a high number of BM blasts correlating with more rapid haematological deterioration and with severe clinical behaviour, cont inues to be applied even today in the prognostic staging system [243]. Greenberg et al., in 1997, established a prognostic score using the multivariate analysis of clinical and biological features resulted by evaluation of 816 patients. In order to calculate a prognostic risk score, the IPSS system uses three clinical factors: percentage of marrow blasts, karyotype, and number of cytopenias (Table 8). Based on this score, the median survival times were 5.7, 3.5, 1.2, 0.4 years for the IPSS risk groups "Good", "Intermediate-1", "Intermediate-2", and "High", respectively [175]. This family consists of over 50 molecules that can be divided into three groups according to their cellular location and their role within this family ( In conclusion, situated at the intersection of many signalling pathways, FAK is a critical tyrosine kinase involved in a variety of processes vital for cellular physiology (Figure 26)

Chapter III-Focal adhesion proteins

[245], [252]. [272], [273].

III-4. FAK in tumorigenesis

III-4.1 FAK levels affect carcinogenesis

The cellular FAK expression is ubiquitous, [START_REF] Weiner | Expression of focal adhesion kinase gene and invasive cancer[END_REF], proved that low levels of FAK mRNA exist in normal tissues, while the primary and metastatic tumours significantly In line with this study, Tavernier-Tardy and colleagues confirm FAK over-expression in CD34 + cells selected from AML patients (with a 65.5% median percentage of positive cells).

In addition, the prognostic analysis confirms the negative impact of this marker for overall patient survival, thus ranking among the phenotypic prognostic markers [292]. The HSP90 dimerization is necessary so that HSP90 may be active and exercise its various functions. It is mainly present as homodimer form α-α or β-β, but there are also heterodimeric forms [298].

III

An interesting observation is the fact that, although HSP90 is abundantly expressed in most cells, only tumour cells contain HSP90 complexes in an activated high-affinity conformations unlike the normal cells where it is in a latent uncomplexated state [297]. This stronger association of client proteins to HSP90 in tumour cells might suggest a protective mechanism involved in its saving from proteasome recycling.

IV-3. HSP90 in tumorigenesis

The HSP90 is a potential target of anticancer therapy due to its involvement in various signalling pathways, cell proliferation, and survival, through regulation of a vast array of client proteins described above (Figure 30

From Campos L. Personal Communication AERES 2010

In AML CD34 + cells, the levels of HSP90 correlated with the percentages of CD34, p170, and bcl-2 positive cells, and its over-expression represents an imprint of poor-prognosis and of resistance to chemotherapy. Elevated levels of HSP90 were also found in samples exhibiting an autonomous growth in liquid culture or which form spontaneous colonies. A concomitant activation of phosphatidylinositol 3-kinase (PI3K) / Akt pathways was observed in these cases [300].

Likewise, in MDS cases with poor prognosis, Duval et al. reported the over-expression of HSP27, HSP60, HSP70, HSP90, and HSP110 mainly in CD34 + cells.

Indeed, the expression of all HSPs, except HSP70, correlates with FAB subtype, IPSS score, Bcl-2 related proteins, CD34, P glycoprotein (Pgp) expression, and with bone marrow blast percentage. All this evidence denotes that their expression could be employed to pursue the disease progression [301].

Significantly higher levels of HSP90 (mean percentage of positive cells (38.3%), FAK (33.8%), phosphorylated FAK (31.4%), accompanied by phosphorylated Akt (26.7%) were traced in mononuclear cells (MNCs) and CD34 + cells from RAEB patients compared with those from RA (7.5%, 4.9%, 3.4% and 5.5%, respectively) or CMML patients (22.1, 19.8, 17.2, and 15.8%, respectively) (p<10 -5 ) [280].

The addition of 17-(Allylamino)-17-demethoxygeldanamycin ), for 24 hours in liquid culture of BM MNCs selected from MDS patients, conduces to the downregulation of HSP90, pFAK and pAKT expression in this cells during the first 12 hours, followed by increased apoptosis at 24 hours [280].

This would indicate that HSP90 over-expression found in refractory anaemia with excess blasts (RAEB) is an unfavourable prognostic factor because it indicates resistance to apoptosis of both normal cells and the blasts.

IV-4. HSP90 as a drug target

The antiproliferative activity of HSP90 inhibitors (radicicol and geldanamycin) is

I. Overview to Experimental Part

To date, the myelodysplastic syndromes are regarded as clonal disorders of haematopoiesis.

Nonetheless, in recent years the role of stroma in etiopathogenesis of these diseases ought to be considered.

Data regarding this topic have been scarce and contradictory so far largely due to the complexity of the network of cells and molecules constituting marrow microenvironment. On the other hand, the scarcity of the date is due to the huge impact of isolation methods on the composition of MSC preparations that cause tremendous differences in the results of different Likewise, the BM microenvironment place ought to be considered as a contributor to drug resistance.

In this respect, the MDS-derived AML is known to be challenging from the therapeutic viewpoint due to the selection of tumour cell clones that express a multi-resistance phenotype and poor response to chemotherapy. The tumour microenvironment might influence drug response and the emergence of drug resistance in both haematological malignancies and solid tumours that metastasize to the bone marrow [224], [306]. Identification of microenvironmental targets, which enable the development of drugs that can be used in association with traditional agents to diminish the environment-mediated drug resistance, may be the response in these cases [224].

The present work aims to clarify some aspects related to adhesion deficiencies of MSCs in MDS settings, as well as their impact on its proliferation rate and on haematopoietic compartment.

II. Materials and Methods

II-1. Patients and healthy donors

Signed institutional review board-approved written informed consent was obtained from all patients and control subjects.

BM aspiates from twelve normal, thirty-five MDS, and three AML-MDS marrow samples were included in the study group.

According Samples were obtained in the absence of any treatment.

Patient diagnosis was performed by cytological evaluation of BM smears after May-Grünwald-Giemsa staining and cytogenetic analysis.

Patients' clinical and biological characteristics are describes in Table 10. supplemented with 20% Fetal calf serum (FCS, GIBCO ® Invitrogen).

0 0 0 0 1 3.5 5 22 0 0 0 0 WHO 2008* RCUD-RA # RARS٭٭ RCUD-RT # RCMD † † † RCUD-RA # RAEB-1 ‡ ‡ RAEB-1 ‡ ‡ RAEB-2 ‡ ‡ AML inv (3)
9) The viability and number of cells were quantified using trypan blue exclusion test.

II-2.2. Lab-Tek Cultures

After sorting, cells were collected in MesenCult ® / FCS (1:1) medium. Cells (1x10 3 per well)

were plated on Lab-Tek ® II Chamber Slides (Nalge Nunc International Corp.) in 0.5 ml MesenCult ® medium and allowed to reach 70-80% confluence over 2 weeks.

II-2.3. Human Haematopoietic Colony-Forming Cell Assays

The detection and counting of erythroid burst-forming units (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) was performed in MethoCult ® H4434 methylcellulose-based medium (StemCell Technologies). For this purpose, mononuclear cells containing the haematopoietic progenitors were separated from BM aspirates using density gradient centrifugation (1.077 g/cm 3 ) on Lymphoprep ™ (FreseniusKabi). After centrifugation, 1.25 x 10 4 BM mononuclear cells were resuspended in 1.2 ml MethoCult ® (StemCell Technologies) and cultured at 37 °C and 5% CO 2 in 35-mm culture dishes (StemCell Technologies) for 14 days. Each sample was processed in duplicate. The colonies were then counted and classified based on morphological features by light microscopy.

II-3. Morphologic and morphometric analysis

II-3.1. Stroma Layers Stain

1. The medium was removed from cultures.

2. The adherent colonies which remain attached to the plate were washed twice using PBS.

3. 5 mL of Methanol (Merck Chemicals Catalog # 106035, Frankfurt, Darmstadt, Germany)

were added by flask for 5 minutes at room temperature (methanol fixes the cells to the tissue culture dishes or flasks).

4. The methanol is discarded and the culture flasks are dried in air, at room temperature.

5. 5 mL of Giemsa Staining Solution (EMD Chemicals Catalog #R03055, USA) are added in each culture flask and leave for 5 minutes.

6. Finally, the Giemsa Staining Solution is removed and the culture flasks are rinsed with distilled water to remove non-bound stain.

7. The tissue culture flasks are allowed to dry at room temperature.

II-3.2. Evaluation of stroma layer composition

The stroma layer composition at 80% confluency was evaluated under an inverted microscope [Inverted (TissueFAXSi ® ) System, TissueGnostics GmbH (Vienna, Austria)] with a magnification x 40.

II-3.3. Morphometrical evaluation of stromal cells from primary cultures

Images were acquired in TIFF format using a PixelLINK PL-A622C/622000227 camera [Aegis Electronic Grup, Inc.], with an x 20 air objective. The field of view's (FOV) with dimensions of 650 x 489 µm, at a resolution of 1600x1200 pixels, were acquired from each flask. These images were then imported into MapInfoProfessional ® 6.5 (Pitney Bowes

Business Insight [formerly MapInfo Corp], USA), in order to perform measurements. Cells and nuclei were measured and values were translated from pixels in µm according to ratio: x [1 pixel / 1 µm in MapInfo] = 0.406 µm microscopically (x value). The calibration of measurements and the equivalence ratio has been established using the square (1 mm) of Burker-Türk camera.

II-4. Flow cytometry

II-4.1. Cell Preparation

The MSCs amplified during 30 to 35 days were harvested using the MesenCult ® Dissociation Kit (StemCell Technologies), collected in glass tubes containing 5 ml MesenCult ® (StemCell Technologies) with 20% FCS and filtered through a 70 μm cell strainer.

The cell number was determined using trypan blue solution (0.4% PBS).

II-4.2. Antibody staining

Cells processed above were pelleted and resuspended in 50 µl washing buffer and stained, on ice for 30 minutes.

Sources and isotypes of antibodies used are detailed in Table 11.

Cell viability was evaluated by staining with 1 µl of Propidium iodide (PI, Sigma, Poole, UK), 1 mg/ml, prior to FACS acquisition.

Data were acquired using a FACS Canto I cytometer and analyzed using DIVA software (Becton Dickinson). We performed immunomagnetic positive sorting using STRO-1 and CD73 marker expression in order to discrimate potential distinct cell subpopulations.

The fraction of STRO-1 + cells was immunodepleted for CD73 by exploiting the differences in epitope density and the avidity of STRO-1 mAb, the STRO-1 + CD73 -cells bearing a higher number of epitope sites (Figure 31). The initial viabilities of the harvested cell fractions, as evaluated by trypan blue exclusion, were 79.6 ± 10.6% for normal stromal cells, 77.6 ± 7.26% for RC stromal cells, and 67.4 ± 10.2% for RAEB stromal cells.

After selection, the STRO-1 + and CD73 + fractions were enriched to >95% (96.34±2.26 for STRO-1 + and 97.74±0.95 for CD73 + cells); percentages were evaluated by cytometry from the singlets gate after excluding dead PI + cells.

Four rounds of selection were performed for each population sorted.

II-6. Immunofluorescence

II-6.1. Cytospin Preparation

Cells remaining after flow-cytometry acquisition were spin down and resuspend at 5000 cells/µl in 4% PBS buffered paraformaldehyde solution containing 10 µg/ml DAPI.

10 µl of this suspension are placed on a glass slide and covered with a coverslip.

Double-stained preparations were visualized under a DM Microscope (Leica, Heidelberg, Germany) at 40 x, and respectively 100 x magnification, using a double band passinterference filter (Omega Optical Inc, Brattleboro, VT).

II-6.2. Culture Slides Preparation

The cells were washed thoroughly to remove residual media and were fixed in paraformaldehyde 4% w/v (Merck Chemicals Catalog, Frankfurt, Darmstadt, Germany) for 10 minutes at room temperature (RT).

II-6.3. Antibody Staining of Slides

Non-specific background staining was blocked with antibody (Ab) buffer containing PBS (1x) / FCS (1% w/v) / BSA (bovine serum albumin, Sigma-Aldrich, St Louis, MO, USA, 0.1% w/v) for 1 h. Cells were then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 20 min before Ab application. Primary antibodies were diluted in Ab buffer before application as follows: FITC-conjugated mouse anti-human STRO-1 mAb (final dilution Before acquisition, nuclear staining with 4' 6-Diamidino-2-phenylindole (DAPI, 1 μg/ml) was performed for 30 minutes at 4 °C.

Slides were mounted in Faramount Aqueous Mounting Medium (Dako Denmark).

II-6.4. Relative Fluorescence Measurements

Triple-stained preparations were visualised under an Axio Observer Z1 microscope (Carl Zeiss, Inc.) at 100X magnification. Signals were recorded simultaneously by three photomultiplier tubes (PMT 1-3). The images (TIFF format) were captured with a PixeLINK PL-A622C/622000227 camera (Aegis Electronic Group, Inc.) by taking multiple exposures through bandpass optical filter sets appropriate for FITC, Texas Red, Alexa Fluor 633 and DAPI using a 100X Plan Apochromat objective.

Analysis and quantification of immunofluorescence staining was performed using ImageJ software (http://rsb.info.nih.gov/ij/).

Five representative fields were analysed per filter and per slide.

Data recorded for an average of 25 cells per group were exported to Excel (Microsoft) for further analysis.

II-6.5. Protein Clustering Analysis

The Origin 7.0 (Microcal Software) charts were used to display the percentage of colocalisation between the analysed proteins in each of 10 spindle-shaped cells and 10 large cells per group. The scatter plots and the colour co-localisation maps were generated by ImageJ software.

The degree of co-localisation was evaluated using the various co-localisation coefficients, and proteins belonging to the same complex were identified using the Intensity Correlation Quotient (ICQ) and intensity correlation plots (ICA) as previously described [307], [308],

[309].

Depending on the value of the coefficient ICQ it can be distinguished 3 patterns of staining:

random staining ICQ~0 (-0,1<ICQ<0,1), segregated staining 0>ICQ≥-0.5, the segregated pattern is assigned to those proteins with the asynchrony or complementary staining (the proteins belonging to different complexes or structures), and dependent staining 0<ICQ≤+0.5. The dependent staining is assigned to the proteins how are the parts of the same complex and their staining intensities should vary in synchrony [307], [309].

II-6.6. Cell cycle and Apoptosis evaluation using DAPI Nuclear Counterstain

Total amount of DNA in cells was obtained by multiplying the number of intensity pixels corresponding to the signal light output of DAPI staining with nuclei area. A histogram stretching was applied then (all pixel values from the ROIs were divided by a constant value) in order to obtain a compact set of values from highly dispersed initial values.

Apoptosis was evaluated by assessing the number of cells containing nuclear changes indicative for this process (cells with irregular edges around the nucleus, chromatin condensation and nuclear fragmentation).

II-6.7. Confocal microscopy

Confocal microscopy was carried out in MSCs cultured on Lab-Tek slides processed as described previously for common fluorescence. Images were acquired using confocal spectral TCS-SP2 microscope (Leica, Heidelberg, Germany).

II-7. Functional Assays

To assess the growth characteristics of the two major MSCs subpopulations, STRO-1 + CD73 - and STRO-1 -CD73 + , proliferation and clonogenicity tests were performed.

II-7.1. Proliferation Tests

To do so, 1x10 3 viable MSCs (quantified using the trypan blue exclusion test) were plated in 25 cm 2 flasks, and the number of cells was counted on days 1, 7, and 14. We then calculated the proliferation index (the difference between the number of harvested cells and the initial plated number) and the doubling time (the duration of one mitosis) estimated by the ratio of the time necessary for 1x10 3 MSCs to reach 80% confluency and the number of population doublings. The number of population doublings was obtained using the following formula: n = log (x/y) / log2, where "x" is the number of initial seeded MSCs and "y" is the cell harvest number [310], [311].

II-7.2. Clonogenicity Tests

The clonogenic potential of MSCs was established with plating efficiency (PE) or CFU efficiency assays. After 14 days of culture, the medium was removed and the colonies were fixed in methanol and treated with Giemsa stain. The colony numbers were then scored. A colony was defined as consisting of at least 50 cells. PE was the ratio of the number of colonies formed to the number of cells seeded x 100% [312].

II-8. Data standardization and statistical interpretation

In the present thesis, we used two types of statistical analysis: descriptive and comparative.

Statistical analysis consisted in determining the arithmetic mean, standard deviation (SD), standard error (ES), and variation coefficients (%). Student's t-test (p) was employed to compare the parameters among the groups and was calculated using SPSS software (SPSS 13.0 Chicago, http://www.spss.com) and Microsoft Excel 2003.

Differences between the groups were considered to be significant when p≤0.05. Briefly the experimental schedule for this article was:

III

1) MSCs isolated from plastic-adherent BM MNCs were amplified until 30-35 days in cultures.

2) Morphometric-morphologic analyses were conducted on stromal cells isolated from MDS vs. normal primary cultures.

3) Thereafter, stromal cells were detached, and two steps of positive selection were performed in order to discriminate the STRO-1 + from CD73 + MSCs.

4) Purity and viability were monitored using flow cytometry and immunofluorescence assays.

5) Cell yield and growth characteristics were evaluated for each fraction and for MSCs selected from MDS compared to normal controls.

6) Statistic analysis were employed to determine correlations between MSC phenotypic abnormalities with MSC growth, and, with haematopoietic compartment dysfunctions (Figure 32). In principle, the RPMI-1640 supplemented with 10% FCS, as well as MesenCult ® media allowed the development of a fibroblastic stromal layer without haematopoietic cells [154],

[313]. MyeloCult ® HT5100 medium allows the development of a stromal layer with haematopoietic progenitors [314].

Cell confluency was scored from 0 to 3, corresponding to a stromal layer covering from < 25% (score 0), 25-50% (score 1), 50-70% (score 2), and > 75% of the area of the culture flask (score 3), respectively.

After four weeks, confluency of adherent layers was observed in all normal cultures, in all systems.

In Refractory cytopenia, 50% of cases (11/22) show deficiencies to reach confluency, particularly in RPMI-1640 medium, but also in MyeloCult ® HT5100:

confluency 0 in two cases,

confluency 1 in six cases, and confluency 2 in three cases (Figure 33). Of note is the fact that in MDS layers exhibited spontaneous lysis in 35 days of cultures, in RPMI-1640 especially.

In RAEB MSC cultures are noticed also:

the colonies architecture disorganization, with clusters of aberrant proliferation, and,

giant "amorphous" deposits in cultures.

Although, prior us, some authors has described these deposits in other haematological malignancies, or in BM metastasis, as fine meshwork of reticular fibres or reticulin / type III collagen, elastin [315], or tenascin [316], their relevance remains obscure. However, the presence of tenascin is regarded as an imprint of pathological states of the bone marrow [316].

The morphometrical evaluation of MSC primary cultures has revealed the different distribution of the three morphotypes of MSC (small, rounded-shaped; thin spindle-shaped;

and large, flat cells) in MDS layers compared with normal settings; with the preponderance of the small-rounded morphotype, on the detriment of large, flat cells, which is considered the onset of terminal differentiation.

III.1-2. BM stromal cells phenotype (MDS vs. Normal controls)

The phenotypic evaluation of MSC layer composition highlights increased percentages of STRO-1 + cells in 20 to 30 days of culture that co-expressed CD106 and CD31 in the RAEB and RC groups. Moreover, this issue persisted in the MDS groups until 60 days, when cell autolysis occurred.

Likewise, under the MDS condition, the CD73 + subsets of MSCs, presents a significant reduction of all studied adhesion markers: VLA 5 or α 5 β 1 integrin (CD49e, CD29), ICAM-1

(intercellular adhesion molecule 1) or CD54, and of extracellular matrix protein (CD44), but the statistical significance was reach only for the CD44, and CD49e.

III.1-3. Growth particularities of BM MSCs selected from MDS vs. normal

The MSC production in STRO-1 + and CD73 + cell cultures from MDS marrows was deficient.

The average proliferation index for MSCs selected from RC marrows after 2 weeks of culture was 17 times lower for STRO-1 fractions and 2.23 times lower for CD73 + , compared with the average MSC output in normal controls. A 6.5-fold drop in STRO-1 + fractions and a 2.4-fold decrease in CD73 + cells were also recorded in the RAEB group.

In addition, the clonogenic ability of the fractions selected from MDS settings was strongly diminished and the differences were more obvious for the STRO-1 + CD73 -cells.

Thus, the MSCs selected from the RC group showed a clonogenic capacity that was 3.5 times lower for STRO-1 + CD73 -, and approximately 2 times lower for STRO-1 -CD73 + compared with normal counterparts. The same decline was noticed for RAEB selected cells compared with normal cells (approximately 4 times lower for STRO-1 + CD73 -fractions and 1.65 times lower for STRO-1 -CD73 + cells, respectively.

In conclusion, the relative proliferation of MDS cultures is the result of a division process that is continuous, but occurs at a low rate and without the ability to generate the normal functional progenitors required to form colonies.

Moreover, a positive relationship was observed between the reduced intensity of CD44 expression in STRO-1 + CD73 + cells in the RC and RAEB groups and the CFU efficiency obtained for CD73 + subsets of cells. In addition, the increased level of CD49e expression noticed in STRO-1 + CD73 + and STRO-1 -CD73 + cells was inversely correlated with the DTs calculated for STRO-1 + and CD73 + fractions sorted from the same RAEB cases samples.

This evidence supports the theory that MSC expansion is an adhesion-dependent process and that CD44 and CD49e molecules are involved in this process.

Likewise, a linear correlation between decreasing CD49e MFIR in MSCs (approximately 2fold) and a 2-fold decline in the number of CFU-GM and BFU-E colonies was also noticed in the haematopoietic compartment of RAEB group.

These observations confirm the haematopoietic growth-supportive role of the MSC layers and

show that this role is α5-integrin-dependent, suggesting that MSCs derived from MDS are functionally incompetent in supporting normal HPC proliferation and differentiation.

Statistical analysis showed that the myeloid expansion is the most affected aspect. In this study, we assessed the expression levels and cellular localisation of focal adhesion (FA) proteins in normal and dysplastic stroma, in order to determine whether their expression correlated with the progression to malignancy.

III.2. Focal adhesion protein abnormalities in

In this order, CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y 397 ], HSP90α/β and p130CAS, and analysed for reactivity, intensity and cellular localization. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y 397 ], and HSP90α/β formed nuclear molecular complexes.

Through these experiments, we found a significant relationship between the levels of expression of FA proteins, their nuclear co-localisation, and the proliferative potential of MSCs.

In addition, the increased levels of pFAK [Y 397 ] in MSCs isolated from MDS patients were inversely correlated with the clonogenicity of haematopoietic progenitor cells (HPCs).

Together, our findings suggest that assessment of the subcellular FA protein co-localisation in MSCs selected from MDS patients in combination with HPC clonality studies are potential tools that could be employed to identify MDS patients with disease progression and to guide therapy. Similar percentages of paxillin positive cells were counted for all three groups.

Cellular distribution of this protein display two patterns of reactivity, diffuse cytosolic and nuclear, with remarkable differences between groups. Thus, 97-98% of RAEB MSCs shows a nuclear pattern of expression for paxillin and a paranuclear staining was revealed by large cells as well. In normal cells, the same cytoplasmic and nuclear disposition was distinguished, but 70% from paxillin positive cells have nuclear expression. In RC group, only 14-15% of paxillin positive cells have also a nuclear reactivity.

Despite the low intensities and diffuse cytoplasmic staining for p130CAS observed in all groups, a slight increase of the number of p130CAS positives cells was found in RC and RAEB groups as compared to the normal group (92%, and 96%, respectively, vs. 73%).

The co-localisation analysis of HSP90α/β with paxillin provides compelling evidence that staining for paxillin varies in synchrony with that for HSP90αβ, consistent with the formation of a complex within restricted, small areas of nuclear regions of 80-90% from RAEB S-MSCs and in 20-30% of normal S cells. Likewise, pFAK [Y 397 ] strongly colocalize at paxillin and HSP90αβ in RAEB cells, while their normal counterparts display a weaker co-localization of pFAK [Y 397 ] to paxillin in approximatively 40 % of studied cells, and no co-localization pFAK [Y 397 ]-HSP90αβ. For RC S-MSCs we noticed very low percents of co-localization between these three proteins (from 0.08% to 0.18%) associated to an exclusive staining pattern.

Moreover, unlike normal L-MSCs, the large cells from RAEB group had preserved an increased level of expression for pFAK [Y 397 ] and a high co-localization of this protein to paxillin and HSP90αβ. The coeficients calculated for their co-localization in RAEB L-MSCs support the fact that the staining intensities vary in synchrony; consequently, these proteins are parts of the same complex (an average of 8% of co-localization was obtained for paxillin-HSP90αβ, 14% for paxillin-pFAK [Y 397 ], and 9% for pFAK [Y 397 ]-HSP90αβ pairs.

The significance of the particular expression of these proteins, as well as their nuclear colocalisation are the subjects of the following chapters.

III.2-2. Role of Paxillin-pFAK [Y 397 ]-HSP90 signalling pathway in MSCs proliferation

Recent evidences have shown that FA proteins, mainly FAK are involved in cell growth signalling pathways [265].

Herein, we bring compelling evidences that strong paxillin nuclear immunoreactivity, and its We have also noticed that increased levels of expression for pFAK [Y 397 ] in MSCs, correlated with a decreased proliferation capacity of HPCs in same patients, i.e., 4-fold lower number of BFU-E and CFU-GM as compared to the normal counterpart, were detected in RAEB group and 1.5-fold in RC group, respectively. This dysfunction detected in the haematopoietic compartment was related rather to the level of pFAK [Y 397 ] expression in MSCs compartment than to the percentage of positive cells for this marker (Figure 36).

This was revealed by the fact that comparable percentages of positive cells in RAEB and RC MSCs cultures (97% vs. 93%) did not have the same impact on the clonogenic ability of haematopoietic precursors. This lies in the differences of pFAK [Y 397 ] intensity of expression between the two groups, which reversely correlated to colony-forming cells (CFC) capacities in haematopoietic compartment (p<0.001, r=-0.60 for RAEB group, and p=0.001, r=-0.31 for RC group, Spearman test). that the percentages of blasts directly correlate with the HSP90, Akt, FAK, and pFAK expression on HPC. Moreover, the patients' survival, as well as the time to transformation into overt leukemia was significantly shorter in MDS cases with higher levels of expression for these proteins in blasts or HPC. These data raises the possibility of using these tests as prognostic tools in MDS. Thereafter we found that 17-AAG (an inhibitor of HSP90)

diminishes the CD34 + and HPC survival in liquid culture, especially in high-risk MDS cases.

Short-term exposure to 17-AAG also down regulates pAKT and pFAK levels. Overall, these data support the implication of this signalling network in MDS and AML pathogenesis and raises the possibility their exploitation as therapeutic target through HSP90 inhibition.

INTRODUCTION

The myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with regard to initial presentation and evolution (1). Patients with MDS usually present with one or several peripheral cytopenias despite a normo-or hypercellular bone marrow (BM). This apparent paradox has been linked to an excessive intramedullary apoptosis (2)(3)(4) but mechanisms underlying this phenomenon are not fully understood yet. We and others have demonstrated that apoptosis results from the activation of caspases, particularly caspase 3 (5,6). The microenvironment is also implicated in the pathogenesis through the secretion of proapoptotic cytokines (Fas and Trail) (7,8). Increased apoptosis is observed in all forms of MDS, but is higher in patients with better prognosis and comparatively lower in patients with an excess of blasts (5,6).

Apoptosis is a tightly regulated phenomenon, and caspase activation is controlled by the bcl-2-family proteins. Indeed we showed that the apoptotic disorder was associated with an imbalance between proteins of the bcl-2 family, with an upregulation of anti-apoptotic proteins bcl-2 and bcl-X L in the forms with excess of blasts (9). More recently the role of Heat Shock Proteins (HSP) in cell protection and apoptosis regulation has been demonstrated.

HSP are a group of highly conserved proteins, which act as molecular chaperones in order to ensure the proper folding of synthesized proteins, or their refolding under denaturating conditions (10). They also play a role in protein degradation via the proteasome machinery. A member of the HSP family, HSP90 is abundantly expressed in the cytoplasm of most human cells. HSP90 exists in two main isoforms: HSP90α, inducible, and HSP90β, constitutive (11).

It exerts its role by forming a multiprotein complex with high ATPase activity, in cooperation with cochaperones, including HSP70 (12). HSP90 clients are implicated in cell cycling, receptor function, signal transduction and apoptosis. High levels of HSP90α protein or HSP90

mRNA have been reported in many types of cancer cells, such as pancreatic carcinomas, breast cancer, ovarian cancer, lung and renal cancer, gastric cancer (reviewed in 13 ).

Furthermore, HSP90 exists mainly in the activated (complexed) form in cancer cells, whereas in non malignant cells only a small part of HSP90 is activated (14).

More limited data are available regarding the expression of HSP90 in haematological malignancies and particularly in acute leukemia and MDS : high expression of HSP90 protein and HSP90α RNA has been reported by Yufu et al in leukemia cell lines and a small series of acute leukemia patients (15). We recently reported on the expression of HSP90 in a larger series of patients with acute myeloid leukaemia (AML) (16). Higher HSP90 levels, as assessed by flow cytometry, were associated with a poor prognosis and with higher expression of activated signal transduction proteins PI3K, AKT and ERK. Other reports show that HSP90 is necessary for the maintenance of oncoproteins such as bcr-abl (17), mutated ckit (18), and flt3 (19,20).

HSP90 activation and functional properties necessitate the binding of ATP to a specific pocket. The benzoquinone ansamycins herbimycin A and geldanamycin are potent inhibitors of HSP90, binding tightly to the ATP pocket and preventing the formation of an active HSP90 complex (21). The less toxic geldanamycin-derivative 17-allylamino-demethoxy geldanamycin (17-AAG) presents a much higher (up to 100-fold) affinity for HSP90 complexes than for uncomplexed HSP90, which confers to this drug a highly specific antitumoral activity (22). 17-AAG (tespimycin) and other HSP90 inhibitors are now considered as targeted therapy for cancer (23).

In a preliminary study, we have shown that HSP27, 70 and 90 are over-expressed in advanced MDS as compared to early MDS and normal BM (24). This suggests their possible implication in MDS pathogenesis and evolution. Here we report on the clinical and biological significance of HSP90 expression in a series of 177 patients with MDS. We evaluated the expression of HSP90 and of relevant client proteins (pAKT, implicated in cell survival and autonomous growth, and pFAK, implicated in tissue invasion and metastasis) at diagnosis and in some cases after evolution to a higher grade MDS or to overt AML. The use of multicolour flow cytometry allowed us to specifically study subsets of cells (ie CD34). We show that HSP90 and FAK are overexpressed in high risk cases, and that CD34+ cells are highly sensitive to the HSP90 inhibitor 17-AAG.

MATERIALS AND METHODS

Patients

One hundred and seventy-seven patients with MDS and chronic myelomonocytic leukemias (CMML) at diagnosis were included into this study between January 2006 and October 2010.

All patients gave an informed consent. Diagnosis was carried out according to WHO recommendations (25) and confirmed by two separate observers. As we included CMML cases, the FAB classification was also used to distinguish sub-groups (26). Cytogenetics was available for 152 cases. Detailed clinical and biological characteristics are given in Table 1.

MDS and control cells

Cells were collected by bone marrow aspiration into heparin-containing vials. Mononuclear cells (MNC) were separated on a Ficoll gradient (Eurobio, Les Ulis, France), washed twice with phosphate-buffered saline (PBS), resuspended in RPMI 1640 (Eurobio) and incubated

for two hours at 37°C on sterile plastic dishes. Non adherent cells were then recovered, washed twice in PBS and immediately processed for further studies.

As controls, normal marrow cells were harvested from 6 healthy bone marrow donors and processed identically.

CD34+ cells

Normal and MDS CD34+ cells were isolated by an immunomagnetic method using the direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec). More than 90% of the isolated cells were CD34-positive (CD34+) after cytometry analysis.

Cultures

Short-term liquid cultures

Non-adherent MNC and CD34+ cells were incubated in RPMI1640 at 37°C in fully humidified atmosphere with 5% CO 2 in the presence of different concentrations of 17 AAG, or of DMSO for controls) for 24 hours. 17-AAG was purchased from Sigma-Aldrich Corp.

(St. Louis, MO), diluted in dimethylsulfoxide (DMSO) and stored at -20°C before use.

After these treatments, cells were washed in PBS and viable cells were enumerated using a trypan blue exclusion test.

All experiments were performed in triplicate.

Clonogenic assays

Normal and MDS/CMML MNC were incubated in triplicate in methylcellulose and growth factor-containing culture medium (STEMα.ID, STEMα, Saint-Clément-les-Places, France). In some experiments cultures were performed in the presence of 17-AAG (or DMSO alone for control) which was added into the culture medium to obtain the appropriate final concentration. CFU-GM and BFU-E were scored after 14 days of incubation.

Antigen expression and flow cytometry

The were matched to the primary antibodies at identical concentrations (27). Cell populations were gated according to CD45/side scatter (SSC) analysis.

Flow cytometry analysis was performed with a Becton-Dickinson FACS Canto II cytometer, using the DIVA software. At least 10000 events were analyzed. Results were expressed as mean fluorescence intensity ratios (MFIR) (ratio of stained sample/control).

Apoptosis

Annexin V staining

Untreated and drug-treated cells were incubated with Annexin-V fluorescein and propidium iodide (PI) (DakoCytomation) in HEPES buffer. After incubation for 15 min in the dark, cells were analyzed by flow cytometry. Live cells were determined by PI exclusion. Early apoptotic fraction was determined by annexin-V-positive and PI-negative stain.

Activated caspase-3 expression

Cytospins were also used to study the caspase-3 activation by Alkaline Phosphatase-Anti Alkaline Phosphatase-(APAAP) technique. We used an amplification combination of alkaline phosphatase (AP) and avidin-biotinyled enzyme complex (ABC) technique (Vectastain Universal mouse and rabbit kit, Vector Laboratories, Burlingame, CA). Cytospins were fixed for 90 seconds with acetone at room temperature. They were rehydrated in PBS for 5 min.

Non-specific binding was blocked with horse serum during 20 minutes. Then slides were incubated for 30 minutes with polyclonal rabbit anti active caspase-3 clone (Cell Signaling, Beverly, MA), washed twice in PBS for 5 minutes and incubated with biotinyled horse antimouse or rabbit secondary antibody for 30 minutes, rinsed again for 5 minutes in PBS, and reincubated for 30 minutes with biotinyled alkaline phosphatase complex (ABC reagent).

After a new step washing in PBS slides were incubated with an appropriate enzyme substrate solution (Fast red) until optimal red granular reaction (20-30 minutes). Slides were rinsed respectively with PBS and distilled water before a counterstain nuclear step consisting of an incubation for 7 minutes with Meyer's Haematoxylin (Dako). After washing in distilled water, PBS, and distilled water again, slides were finally mounted in Fluorotech aqueous media (Valbiotech, Paris, France). Controls were performed by replacing the primary rabbit antibody by an irrelevant antibody of the same isotype. Slides were examined using X10 magnification by two observers. Cells were considered stained if any diffuse reddish cytoplasmic staining could be identified. A scale of three levels of red staining was used to assess intensity of staining : 0 for no staining, 1 for weak staining, 2 for strong staining and 3

for very strong staining. The percentage of positive cells (strong or very strong staining) was determined after counting 100 cells.

Statistical analysis

Mann-Whitney (or Kruskall-Wallis) non parametric tests were used to compare the means of two (or more) groups. Proportions were compared by Chi-square test (or Fisher's test when a group comprised less than 10 units). Correlations were performed using a Spearman rank correlation test.

Survival curves were plotted according to the Kaplan-Meir method. Survival duration of different groups was compared by the log-rank test. Multivariate analysis of survival was performed using a Cox regression model.

Statistical tests were computed by the IBM SPSS statistical software and data plots were performed using the Prim5 software.

RESULTS

Expression of HSP90, FAK, pFAK and pAKT

Expression of HSP90 was weak in MNC and in the "blast" gate (CD45 dim /SSC low ) of normal marrow cells. We also observed a low expression of FAK and pFAK. pAKT was not detected above control level in normal MNC. Results were similar in normal CD34+ cells for all proteins studied.

In MDS MNC cells HSP90 and other protein levels were significantly higher in high-risk cases according to FAB classification (Fig. 1). When cases were stratified according to WHO classification, the expression was higher in RAEB-I + RAEB-II (pooled together) versus other forms. The expression of HSP90 was also higher in RAEB-II vs RAEB-I (p < 0.05) and there was a trend for higher levels in RCMD versus RA (with or without ringed sideroblasts) (p=0.06). Similar results were obtained considering percent positive cells instead of MFIR (data not presented). There were also significant differences regarding cytogenetics subgroups. HSP90, FAK, and pFAK levels were lower in good prognosis than in intermediate and poor prognosis forms (fig. 2). pFAK levels were also more elevated in poor prognosis forms than in intermediate. The same differences were observed in CD34+ cells, but, in addition pAKT levels were also different in god versus intermediate or poor prognosis groups.

5q-cases expressed similar levels as other good prognosis cases. Finally, although these proteins were not detected only in the "blast" gate (CD45/SSC low) of MNC cells, we observed a weak linear correlation between the percentage of blasts (as assessed by cytology or by cytometry) and the expression of HSP90, pAKT, FAK and pFAK (r2=0.62 to 0.71).

These proteins were also expressed at significantly higher levels in CD34+ cells than in CD34-negative MNC (p<10 -4 for all proteins). We therefore compared the level of expression in CD34+ cells in the different sub-types of MDS and in CMML. Again, we observed a higher expression of HSP90, pAKT, FAK and pFAK in RAEB than in RA/RAS, CMML exhibiting intermediate levels (Figure 1). This shows that the differences regarding expression in MNC cells were not only due to the higher percentage of blasts or CD34+ cells in high-risk cases.

Predictive value

Because of the correlation with other relevant clinical factors such as cytogenetics and percentage of blasts we studied the prognostic value of HSP90, pAKT, FAK and pKAK expression. For univariate analysis, patients were arbitrarily placed into two categories : high level (MFIR equal to or above median value) or low level (MFIR below median value).

DISCUSSION

In this paper, we show that high expression of HSP90 is associated with more advanced disease and worse clinical prognosis. As expected, this was significantly correlated with other known prognostic features such as cytogenetics and blast percentage which appear to be of importance in multivariate analysis. One interesting point is the fact that CD34+ cells from advanced cases also expressed the higher levels, when compared to CD34+ cells of good prognosis cases or normal marrows. This implies that progression to advanced disease is associated with a distinct transduction pathways profile at the CD34+ progenitor level, where an imbalance between proliferation and apoptosis has already been described (28). In line with this observation, we also show that when patients could be retested at the time of transformation into overt leukemia, the expression of HSPs ang signaling molecules was significantly increased. This is also consistent with the already known overexpression of HSP90 in acute myeloid leukemia (15,16).

We also studied the expression of the activated forms of known clients of HSP90, pAKT and pFAK. These proteins are implicated in signal transduction and their constitutive activation is a hallmark of transformation in many cancer models. Again a higher expression was observed in MNC and in CD34+ cells of MDS with poor prognosis or adverse cytogenetics. Indeed high expression of HSP, FAK and pAKT was associated with higher risk of transformation and poor survival. High levels of HSP and FAK are predictive of resistance to chemotherapy in AML as shown by our group (16) and others (29). More controversial data exist regarding the prognostic significance of pAKT activation in AML cells. While we (16) and others (30) have observed a poor prognosis, Bardet et al showed that pAKT expression as detected by flow cytometry implied a better prognosis, that was observed independently of cytogenetics, although pAKT was higher in CBF and intermediate cases (31). These discrepancies may be the consequence of different technical approaches, but mainly due to the fact that different sites of phosphorylation were assessed in these studies. Again, we show that levels in CD34+ cells were higher at time of evolution to AML than at diagnosis, irrespective of the initial classification of MDS. Taken together, these findings favor a role for these proteins in disease initial type and in the pathogeny of evolution into overt AML.

The second point raised in our study is the possibility to target in vitro HSP90. HSP90 inhibitors such as geldanamycin, 17-AAG or 17-DMAG have anti-tumoral activity by inhibiting proliferation and inducing apoptosis (32,33). In leukemia cells17-AAG alone or in combination with chemotherapy inhibits cell growth and induces apoptosis (34). Moreover, in specific leukemia subtypes characterized by the presence of mutations or rearrangements of genes such as flt-3 or bcr-abl resulting in high expression of constitutively activated oncoproteins, the association of HSP90 inhibitors and targeted drugs is highly effective invitro (35)(36). Although the exact mechanism by which HSP90 inhibitors interfere with leukemia cell survival is not fully understood, we have also demonstrated that 17-AAG was able to induce apoptosis in primary AML cells, in correlation with HSP90 and activated AKT levels (16). In MDS, we show that 17-AAG readily inhibits CD34+ and MNC cells survival in liquid culture, at least in samples from high grade MDS. Short-term exposure to 17-AAG also down regulates pAKT and pFAK levels. This is consistent with the mechanism 17-AAGinduced apoptosis suggested by Nimmanapalli et al, implicating a modulation of apoptotic proteins of the bcl-2 family downstream of Akt, Raf-1 and Src down-regulation (37). We Epigenetic therapies are increasingly used in MDS. The HDAC inhibitors show a clinical activity in high-grade MDS. Acetylation of HSP90 by exposure to histone-deacetylase (HDAC)-6 inhibitors results in an inhibition of its chaperone function (38). The combination of an inhibitor of HDAC and 17-AAG is highly active in-vitro against cells ofchronic myeloid leukemia in blast crisis and AML cells harboring a mutation of flt-3 (39). HDAC6 inhibitors, alone or in combination with another HSP90 inhibitor, may represent a potential targeted therapy of high risk MDS with possible dual mechanism of action.

MFIR

GENERAL DISCUSSION

The present work is dedicated to the argumentation of the morpho-molecular roles of mesenchymal stromal cells (MSCs) in myelodysplastic syndromes (MDS) pathogenesis.

Myelodysplastic syndromes represent a heterogeneous group of myeloid neoplasms in terms of severity, prognostic, progression to acute myeloid leukemia (AML) and therapeutic approach solutions.

The evidence accumulated in recent years significantly improved the understanding of the pathogenic mechanisms responsible for the selection of neoplastic hematopoietic clone(s), resistant to apoptosis and able to express factors promoting its own growth, proliferation and migration.

The understanding the mechanisms by which neoplasia manages to integrate the stromal components in tumorigenesis process represents also a major source of progress. The haematopoietic stem cells (HSC)-MSC relationship is a critical point in the haematopoietic malignancies pathogenesis, and the current technical approaches provide limited and rather slow progresses.

The complexity of our subject required the integration of the knowledge of several areas: haematologic oncology, cytology, immunology and molecular biology, that along with our strongly motivation for the subject constituted a solid foundation for developing and promoting this doctoral theme.

Preliminary hypothesis was that the MSCs, the genitors of the all stromal cells have a pathologic behaviour in MDS and are unable to generate a microenvironment suitable to haematopoietic cells development.

This project establishes the immunophenotypic profile and the growth patterns of MSCs from MDS microenvironment associated to the malignant haematopoietic clone, throughout of the MDS disease evolution to acute leukemia, highlighting the distortion aspects related to adhesion processes, such as cell proliferation, clonogenic growth and neoangiogenesis.

With our results we want to extend the current level of knowledge in the MDS field by decoding the signalling pathways involving the focal adhesion proteins with impact in neoplasia dynamics and to inventory the common or distinctive elements of the tumoursupport system relationship in order to identify the discreet pathogenic models which could be employed as therapeutical targets.

1. Myelodysplasia could be also a disease of stroma?

The MDS pathophysiology remains a highly controversial topic.

To date the MDS is generally regarded as clonal disorders of haematopoiesis; the disease origin is thought to be in a genetic transformation of haematopoietic stem / progenitor cells.

However new insights reevaluates the microenvironment place from indifferent spectator to principal actor.

Thereby, there is the histopathologic evidence of the microenvironment disruption in MDS.

In There are some research teams which attribute the MSC failure in primary cultures from MDS on account to the aging mechanisms of (stem) cell population(s) during long-term cultures, that induce an decreasing ability to self-renew and to properly differentiation [319]. Our evidences are not consistent with this hypothesis because the normal MSC control does not reach these changes during the 30 days of cultures.

In addition, we have arguments that MSC cultures from RAEB fulfill the "dysplasia" criteria requested by histopathologic term, which has been definited as "a loss in the uniformity of the individual cells, as well as a loss in their architectural orientation" [320]. Thus, the RAEB MSC cultures were characterized by a higher rate and disordered proliferation (which no longer comply with so-called "the cohesive zone formulation" or "cell-substrate contact areas", that are formed predominantly by larger cells in the middle and are surrounded by a cuff of spindle-shape cells to the periphery) [134], [321] reflected in the microscopic image by colonies architecture disorganization with clusters of aberrant proliferation.

Moreover, it seems that these cells have an intense metabolic activity transposed into giant "amorphous" deposits in cultures. All these evidences are consitent with hypothesis that myelodysplastic stroma environment along with intrinsic changes in a hematopoietic stem/progenitor cell clone might equally contribute to the abnormal hematopoiesis in MDS.

The phenotypic characterization of the MSC niche in in vitro primary cultures

In order to identify the potential phenotypic abnormalities of MSC in MDS settings, our study begins with normal MSC cultures assessment. The flow-cytometric approach of MSCs harvested from in vitro primary cultures allows discrimination of three types of cells that match in terms of size and granularity those identified by morphological and morphometrical evaluation. Moreover, supplementary staining with two MSC specific markers, STRO-1 and CD73, complements and supports previous data, i.e., in these cultures there are three cellular phenotypes: STRO-1 + CD73 -, STRO-1 + CD73 + , and STRO-1 -CD73 + .

The STRO-1 + cells were CD45 low , expressed the vascular endothelial-related markers, CD106

(VCAM-1) and CD31 (PECAM-1), and had decreased levels of the adhesion markers CD44, CD54, CD29, and CD49e.

This particular expression for the two endothelial-related markers, CD106 and CD31 raised the question if they are not the imprint of a population with an anatomical distinct location in BM: pericytes which coexist with mesenchymal cells in our cultures? Or their association with the STRO-1 molecule it does not mean the fact that this is a potential MSC subpopulation, more immature in MSC developmental hierarchical tree?

The first hypothesis is sustained by Bianco P et al. in 2001, andShort B et al. in 2003 that interprets the CD106 expression, previously reported in umbilical cord blood (UCB) and BMderived MSCs [323], [324], as the imprint of a particular location (the nearby outer surfaces of blood vessels) and may share an identity with the vascular pericytes [START_REF] Bianco | Bone marrow stromal stem cells: Nature, biology, and potential applications[END_REF], [START_REF] Short | Mesenchymal Stem Cells -Review Article[END_REF]. Moreover, this hypothesis is supported by the co-expression of α smooth muscle actin or 3G5 antigen on these cells, which is recognized as a specific marker for pericytes [START_REF] Gronthos | Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[END_REF].

The second hypothesis is supported by [START_REF] Shi | Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp[END_REF], which described a minor subpopulation of STRO +hi VCAM-1 + cells isolated from freshly BM, possessing CFU-F forming capacity, which appears to be a non-cycling population in vivo, because the Ki-67 expression lack on this minor subpopulation, exhibits telomerase activity, and shows an undifferentiated phenotype and substantial proliferation in vitro [START_REF] Gronthos | Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[END_REF]. Moreover, these highly proliferative clones derived from single STRO-1 +hi VCAM-1 + cells exhibited osteogenic, chondrogenic and adipogenic cell differentiation in vitro [START_REF] Gronthos | Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow[END_REF]. Their unlimited potential for division and proliferation is also supported by observations that the small number of STRO-1 + cells seen in cultures at later points were able to produce adherent cell layers with the same cellular composition and phenotype as those generated by STRO + cells freshly isolated from BM [123].

Likewise, as previously reported by [START_REF] Simmons | Isolation, characterization and functional activity of human marrow stromal progenitors in hemopoiesis[END_REF]Gronthos S et al. in 1996 [127], [128], we found that the STRO-1 + cells express CD31, too, and possible phenotypic pitfalls, which may contribute to the tremendous differences between different groups is the fact that PECAM is sensitive to trypsin [308].

induced by TNFα stimulation [321]; high levels of TNFα are common in MDS [190], and MSCs themselves could be responsible for its synthesis in the absence of HPC stimulation.

The role of this cytokine in the MDS microenvironment is probably related to its capacity to induce internal proliferative signals in MSC cells, as previously noted by Kohase et al. [308].

The latter is related to CD106 function as a major ligand for selective CD29-mediated HPCto-MSC adhesions, and thus, its influence on the HPC mitotic rate and division kinetics [309].

The increased expression of CD31 + could be an imprint of the neoformation of blood vessels in MDS settings, as Boudard D et al. showed in a previous study [190].

In addition, in MDS settings, the CD73 + fractions of MSC display a significant reduction of adhesion markers, CD29, CD54, CD44, and CD49e.

MSC growth deficiencies in MDS are directly correlated with diminution of adhesion markers expression:

Furthermore, the functional tests revealed MSC growth abnormalities in the absence of any contact with or stimulation by soluble molecules from HPCs and proved the pathological nature of stromal precursors in MDS settings.

Thus, MSC production in STRO-1 + and CD73 + cell cultures from refractory cytopenia with unilineage dysplasia (RCUD) and refractory dysplasia with multilineage dysplasia (RCMD) marrows was deficient, and, in addition, the clonogenic ability of these fractions was strongly diminished. We conclude that the relative proliferation in MSC cultures from RCUD and RCMD is the result of a continuous division process occurring at a low rate and lacking the ability to generate the normal functional progenitors required to form colonies. By contrast, in refractory anaemia with excess of blasts (RAEB) settings, the proliferation rate is moderately improved due to the reduced doubling time of STRO-1 cells. However, this was not accompanied, at the end point, by complete functional maturity as reflected in the CFU-F number.

Likewise, the diminution of CFU-F capacity of CD73 + fractions in MDS settings that directly correlates with the CD44 mitigate on their surface should be pointed out. In addition, the doubling time of MSCs from MDS inversely correlate with their expression for CD49e. In conclusion, the MSC growth defects were significantly correlated with decreases in CD44 and CD49e (α5-integrin) adhesion molecules.

Molecular mechanisms whereby pFAK expression can influence the clonogenicity of haematopoietic precursors (HPC)

Another hypothesis that arises from our study is the putative role of FAK [Y 397 ] expression on MSCs in HPC-to-MSC interactions and thus its implication in modulating HPC clonogenic capacities (Figure 38). or presence of an anti-α 5 -integrin blocking antibody, as well as conducting tests to evaluate cell division symmetry and LTC-IC assays.

Which is the echo of these results in diagnostic customization in MDS?

Correlations between MSCs abnormalities (i.e., adhesion deficiencies, growth dysfunctions, abnormal expression of FA proteins) with MDS prognosis and resistance to chemotherapy could lead to a new classification of MDS cases into risk groups involving microenvironment factors.

CONCLUDING REMARKS

1. Our results reinforce the concept "bad seeds in bad soil" that underlies the neoplastic evolution in MDS settings.

2. Herein we bring arguments that MSCs isolated from MDS have a pathological behaviour in primary and secondary cultures and that they actively contribute in some neoplasic signalling pathways involving the adhesion-related processes, such as cell proliferation, apoptosis, clonogenic growth and neoangiogenesis.

3. Structural and functional analysis allowed us the identification of morpho-and immunophentotypes that could be involved in the conflict between HSC -"adhesive" microenvironment, proposing also an in vitro study model of the relationship between these two partners.

I

  owe my deepest gratitude to Prof. Dr. Denis Guyotat for the interesting conversations, detailed review and for thoughtful comments. Thanks to all the people who have made the time I have spent in Saint Etienne fun and memorable. It has been great! Many thanks for keeping me sane and on the right track! Special thanks go out to Annie for her assistance with cell cultures and immunofluorescence staining, to Salima and Caroline for cytometry assistance, to Department of Genetics, Nathalie, Béatrice, Jacques, and Sylviane for their assistance in fluorescence microscopy and FISH technique, and to BioMol Department, Eliane (for the folk dances), Elisabeth, and Françoise for their help in extraction and quantification of DNA, RNA. Thanks also to all biologists of Haematology Lab (CHU St Etienne), Christian Vasselon, Pascale Flandrin-Gresta, Nathalie Nadal, Françoise Solly for the interesting scientific discussions that we had. Not at least, I would like to thank to Maryse for her patience and for the enormous amount of work, which she made with administrative papers and reagents orders. ii I am also pleased to acknowledge the expertise of Jean-François Mayol in the MSC functional assays and of Claude Lambert in cell sorting. I would also like to thank to Professor Eugen Carasevici and to the other members of Haematology and Immunology laboratories, "St Spiridon" Hospital, Iasi because they gave me the opportunity to work and to learn with them. Specially thanks to Dr. Florin Zugun-Eloae for him patience, interesting conversations and technical support. I would like to thank to members of the Service Hématologie Clinique, Institut de Cancérologie de la Loire, and to Haematology Department of Saint Spiridon Hospital, for their constant assistance with cells prelevements and for their permanent enthusiasm for science.

Figure 6 . 7 . 8 . 9 .Figure 19 .Figure 34 .Figure 35 .Figure 37 .Figure 38 .

 67891934353738 FIGURES

Figure 1 .I

 1 Figure 1. Schematic representation of stem cell hierarchical tree iPS: induced Pluripotent Stem cells; HSC: haematopoietic stem cells; CLP: common lymphoid progenitors; CMP: common myeloid progenitors; GMP: granulocyte / macrophage progenitor; MEP: megakaryocyte / erythroid progenitor; MSC: mesenchymal stromal cells. Adapted from: Wagers A. J. & Weissman I. L., Cell 2004, and Junying et al, Science 2007

Figure 4 .

 4 Figure 4. Schematic diagram depicting potential mechanisms involved in adult HSC-NSC plasticity. Adapted from: Wagers A. J. & Weissman I. L., Cell 2004

Figure 5 .

 5 Figure 5. Transcription factors implicated in haematopoietic lineage development LMO2: LIM-only protein, E2A: Ebox binding protein, SCL: stem cell leukaemia, HEB: HeLa Ebox Binding protein, EBF: early B cell factor, GATA-1/2/3: GATA binding protein, C/EBP/α and β: CAAT/enhancer binding protein, Pax5: paired domain protein, FOG: Friend of GATA, From: Hoang T., Oncogene 2004

Figure 6 .

 6 Figure 6. Extrinsic factors implicated in the fate of HSC and other haematopoietic progenitors Wnt: Wnt signalling protein, Dl, Ser: Delta, serrate (Notch ligands), BMP: bone morphogenic protein, Shh: Sonic Hedgehog, VEGF: Vascular endothelial growth factor, SF: Steel factor, KL: Kit ligand, SCF: Stem cell factor, LIF: Leukaemia inhibitory factor, GM-CSF: Granulocyte/macrophage colony stimulating factor, G-CSF: Granulocyte colony stimulating factor, M-CSF: Macrophage colony stimulating factor, Tpo: Thrombopoietin, Epo: Erythropoietin. From: Hoang T., Oncogene 2004

Figure 8 .

 8 Figure 8. Effects of DNA methylation on gene expression From: Ling C. & Groop L., Diabetes 2009

Figure 9 .

 9 Figure 9. Human haematopoietic CFC assays in MethoCult ® media: Procedure diagram From: Human Colony-Forming Cell Assay Using MethoCult ® , Technical Manual, Stem Cell Technologies, Catalog #28404, 2004

Figure 11 .

 11 Figure 11. Representative image for CAFC assaysCobblestone area-forming cell (CAFC) integrates tightly in the adherent layer where they cyclically proliferate and release their progeny during8-10 weeks (Magnification, x 1000)

Figure 12 .

 12 Figure 12. Histology of the bone marrow Osteoc: osteoclasts, hemat c: haematopoietic cells, barrier c: barrier cells, blc: bone-lining cells, osteobl: osteoblasts, adipo: adipocytes, advent c: adventitial reticular cells, eryth isl: erythroblastic islands, meg: megakaryocytes. From: Weiss L. & Geduldig U., Blood 1991

Figure 13 .

 13 Figure 13. Morphological similarities between BM MSCs with long projections and ARCs CD10 immunostaining of BM trephine biopsy. BM MSCs: bone marrow mesenchymal stromal cells, ARCs: adventitial reticular cells, BLCs: bone lining cells, P: pericytes. From: Jones E. and McGonagle D., Rheumatology 2008

Figure 14 .

 14 Figure 14. Model of adipocyte differentiation from ARCs.

Figure 15 .

 15 Figure 15. Bone marrow stromal cells Wright-Giemsa Stain of bone marrow osteomedullary biopsy. A) Adipocyte (Magnification x 400), B) Endothelial cell (Magnification x 600), C) Fibroblast (Magnification x 600), D) Osteoblasts (Magnification x 400), E) Osteoclast (Magnification x 1000), F) Macrophage (Magnification x 600)

  that ECM is part of the BM microenvironmental niche, being involved in regulation of the balance between replication and differentiation of MSCs in response to appropriate signals [101]. Several mechanisms are supposed to facilitate the expansion of marrow-derived MSCs and prevent their differentiation (i.e. into osteoblasts): -ECM modulates the activity of growth factors by controlling proteolytic activation of latent factors (i.e., transforming growth factor-β [TGF-β]); -ECM interacts with cell surface receptors preventing the cognate ligand binding (i.e., as in the case of epidermal growth factor (EGF) receptor), or by sequestration of differentiation factors such as platelet-derived growth factor (PDGF) and bone morphogenetic proteins (BMPs), or of Wnt proteins (which are involved in MSC differentiation through LRP5 and LRP6 binding the glycosaminoglycans of the ECM); -ECM may also bind the growth-promoting factors from the serum; -and, ECM could enhance the function of putative accessory cells that support MSCs replication [101]. There is evidence that denatured collagen type I (DC) matrix promotes the maintenance of in vitro osteogenic differentiation of MSC by influencing the retention of early osteogenic functions in the late passage cells during ex vivo expansion [102]. The same ability to preserve MSC adipogenic potential during the late passage of MSCs ex vivo, expanded on the DC matrix in contrast with the MSCs expanded in culture plastic tissue was demonstrated by Mauney J. R. et al. in 2005 [103]. The mechanism most likely involved in influencing the ability of the DC matrix to promote retention of adipogenic differentiation potential of MSCs in vitro is the maintenance of certain replicative ageing-functions (i.e., cell proliferation capacity, inducibility of stress-protective proteins) [103].

  3.1. Terminology Over the time, numerous terms were assigned to describe the adherent cells derived from many adult tissue sources displaying fibroblast-like morphology: precursors of nonhaematopoietic tissue, colony forming unit-fibroblast (CFU-F), bone marrow stromal [stem] cells [BMSCs] and/or stromal precursors cells (SPCs), RS-1, RS-2, mMSCs (RS: recycling stem cells) (m: mature), multipotent adult progenitor cells (MAPCs) [104], fibrocytic cells, fibrocyte, fibrocytic colony, fibroblast, fibroblast colony, precursors for fibroblast colonies, fibroblast-like colonies, stellate colonies, fibroblastoid stromal cells, stromal cells, CD34 -/low haematopoietic stem cell clones with mesenchymal stem cell characteristics, circulating skeletal stem cells, CD34 -CD105 + mesenchymal cell lines, CD34-negative fibroblast-like cell lines, CD34-negative CD105-positive cell line, and mesenchymal stem cells [105], to enumerate just some of them.

Figure 16 .

 16 Figure 16. MSCs morphology and morphometry

  specific features for MSCs. The second population of cells was smaller than 3 µm, polygonal in shape and had limited renewal capacity[116]. Finally,Staszkiewicz et al. found that the size of ear-derived MSCs ranged from 8.1 to 26.6 µm in diameter, with the majority of the population (~72%) between12 and 20 µm [117]. The common opinion is that human CFU-F are positive for vimentin[START_REF] Da | In Search of the In Vivo Identity of Mesenchymal Stem Cells[END_REF], [105], [118], [119], [120], type I and type III collagen [ 105], [121], [122], type IV collagen [105], [122], fibronectin [105], [118], [120], [122], fibrin [105], [118], acid phosphatase [105], [121], [122], α-Naphthyl-acetate esterase, α-Naphthyl-butyrate esterase [105], [121], periodic acid Schiff (PAS) [105], [122], Sudan Black [105], [122]. Likewise, part of MSCs from primary cultures express smooth muscle (sm) α-actin [105], [120]. I-3.3.4. Phenotype To date, there is no consensus regarding a single surface antigen that could identify the MSCs. For this reason, a panel of several positive and negative markers should be employed to characterize these cells. The International Society for Cellular Therapy proposed for MSC characterization three positive markers. i.e., CD73 (ecto-5'-nucleotidase, SH3, SH4), CD105 (endoglin, SH2), and CD90, and the absence of expression for haematopoietic related markers such us CD45, CD34, CD14, and CD11b [106].

  authors have reported that CFU-Fs are CD45 med/low [120],[124]. Moreover, there is evidence that freshly isolated MSCs display the greatest percentage of CD45-positive cells, whereas MSCs expanded in culture over more passages almost completely lose their expression of this marker [117],[125].Regarding the CD45 functions in non-hematopoietic cells, this molecule serves as a negative modulator of growth factor receptor tyrosine kinases, in addition to its well-established role as an activator of tyrosine kinases src family[126].Endothelial-related markers have also been reported to be expressed by MSCs, although these markers excite also controversy: PECAM-1 (CD31), P-selectin (CD62P), factor VIII-related antigen, and thrombomodulin [127],[128]. In terms of HLA-molecules, MSCs express HLA-1, and do not express HLA-DR[120].

5 .

 5 Schematic model depicting mesenchymal stromal cell proliferation and differentiation The MSCs are generally thought to be resident in the perivascular compartment of many tissues, in fat, skin, muscle, and other locations [130], [131]. Their proliferation process involves two distinct cellular compartments. In the first compartment, MSCs undergo transcriptional modification, generating precursor cells without apparent changes in phenotype and self-renewal capacity. This process especially interests the MSCs residing in adult bone marrow, which are quiescent and growth arrested in G 0 /G 1 outside stimulation with growth factors. In vitro, upon stimulation, the multipotent, uncommitted MSCs undergo asymmetric division, giving rise to two daughter cells, one being the exact replica of the mother cell and maintaining multilineage potential, and the other daughter cell becoming a precursor cell, with a more restricted developmental programme. In this model, the precursor cell continues to divide symmetrically, generating more tripotent and bipotent precursor cells. These tripotent and bipotent precursor cells are morphologically similar to the multipotent MSCs, but differ in their gene transcription repertoire, and therefore, still reside in the stem cell compartment. The progression of MSCs to precursor cells is considered the first step in stem cell commitment. The transition or exit from the 'stem cell compartment' to the 'commitment compartment' occurs when precursor cells continue to divide symmetrically to generate unipotent progenitor cells, simultaneous with the acquisition of lineage specific properties, rendering them fully committed mature cells with distinguishable phenotypes [104].

  neuronal cell adhesion molecule (NRCAM), FK506 binding protein 5 (FKBP5), interleukin 1 type II receptor (IL1R2), zinc finger protein 145 (ZNF145), tissue inhibitor of metalloproteinase 4 (TIMP4), and serum amyloid A2. The function of these genes cover a broad range of cellular processes, including cell adhesion, protein folding, organization of actin cytoskeleton, as well as inflammatory response[104].The lineage committed cells of MSCs can fabricate a spectrum of specialized mesenchymal tissues including bone, cartilage, muscle, marrow stroma, tendon, ligament, fat (Figure17) and a variety of other connective tissues[131],[132].

Figure 17 .

 17 Figure 17. Schematic model of mesengenic process The stepwise cellular transitions from the putative MSC to highly differentiated phenotypes.From: Caplan A. I. and Bruder S. P., Trends in Molecular Medicine 2001

  bodies under low oxygen, and which keep the differentiation potential into neural cells are considered to be the nearest of embryonic stem (ES) cells[134]. It includes very small embryonic-like (VSEL) stem cells, which were isolated from murine bone marrow by positive selection using the chemokine receptor CXCR4, and the multipotent adult progenitor cells (MAPCs) isolated from human bone marrow under low oxygen and low serum [134]. These cells express genes such as Oct-4 (characteristic of ES) and Dickkopf-1 (DKK-1, an inhibitor of the canonical Wnt signalling pathway) [134]. Recently, L Basciano et al. showed that in vitro exposure of MSC to hypoxia enhanced (4-60 fold) the expression of the genes involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, LAMA), and epithelial development (DMKN). In conclusion, hypoxia maintains the cells undifferentiated and, in parallel, enhances the expression of genes involved in the development of various, mesodermal and non-mesodermal cell lineages. In this respect, hypoxia may increase both the multipotency and the trans-differentiation potential (plasticity) of MSC [135]. I-3.3.7. MSCs create their own niches in vitro cultures During expansion on cultures, MSCs undergo different changes and create an in vitro niche. Within this niche, cells play different roles. Thus, Prockop DJ noticed in early phases of MSC cultures a population of spindle-shaped cells (type 1, or RS-MSCs) that express surface proteins with an inhibitory influence on cell adhesion [such as α 6 -integrin and podocalyxinlike protein (PODXL)]. These cells are highly motile, secrete DKK-1, and serve as nurse cells for other subpopulations; hence, they are key elements of the rapid growth phase [134]. As the colonies expand, secretion of Dkk-1 decreases and expression of PODXL and the related proteins are lost. The colonies then enter a near stationary or plateau phase, as the colonies become more tightly packed and develop distinct inner and outer regions [134], [136]. The inner regions are populated with the more commitment precursors, but this stage is readily reversible, in that replating either the inner or outer regions generates single cell-derived colonies with the same characteristics as the initial colonies [134], [136]. At each replating of a colony at clonal density, the daughter colonies are heterogeneous in both size and their potential to differentiate as is represented in Figure 18 [134], [137], [138]. The reversibility of the cultures, however, markedly decreases if the colonies are allowed to expand to confluency, in that there is a dramatic decrease in clonogenicity (from 90% CFUs-F to <20%), and also stands out a decrease in the potential for multilineage differentiation, and increased expression of epitopes such as STRO-1 and GD-2 [134], [139]. However, even in confluent cultures resulting after several passages, a fraction of the cells remain clonogenic, suggesting that there is persistence of one or more in vitro niches containing different subpopulations [134].

Figure 18 .

 18 Figure 18. Schematic model depicting the changing properties of human MSCs expanded in culture. From: Prockop DJ, Molecular Therapy 2009

3 . 6 .

 36 Photographs of representative CFU-F-derived colonies are shown in Section 3.3. Ensure that there is a linear relationship between the cell numbers plated and the resulting colony numbers, by confirming that there are twice as many colonies when 2 x 10 6 cells are plated as compared to 1.0 x 10 6 cells. Likewise, there should be twice as many colonies when 1.0 x 10 6 cells are plated as compared to 0.5 x 10 6 cells. Ideally there should be 10 -40 colonies per 100 mm dish or T-25 cm 2 flask. Linearity may not be observed outside this range, as the cells would have been under or overplated.Each bone marrow sample is unique for that donor and the number of CFU-F may depend on a number of factors including age, presence of disease, and previous treatments given to the patient[154].

Figure 19 .

 19 Figure 19. Multilineage differentiation pontential of MSCs A) von Kossa stain of mineralized deposits in MSCs cultures indicate osteogenic differentiation (Magnification x 200); B) Oil red-O staining indicate the presence of lipids in adipocyte cells within two weeks of adipogenic induction (Magnification x 200); C) Aggregate of collagen in cultures indicate chondrogenic differentiation (Magnification x 200). From: Gronthos et al., Journal of Cell Science 2003

(

  JAG1) expression, which signals through Notch receptors on adjacent HSCs promoting their self-renewal [156]. Conversely, bone morphogenetic protein (BMP) binds to BMP receptors A and B expressed by osteoblasts, and promotes quiescence of HSCs. Likewise, osteoblasts synthesize Dkk1 (an inhibitor of Wnt signalling), and angiopoietin 1 (ANG-1), which binds to receptor tyrosine kinase TIE2 expressed on HSCs promoting HSC maintenance in the bone marrow [44], [156]. Moreover, the osteoblasts express the vascular cell adhesion molecule 1 (VCAM-1) which contributes to HSCs immobilization, protect from apoptosis and promote quiescence (Figure 20) [156].

Figure 20 .

 20 Figure 20. Molecular interactions between HSCs and osteoblasts at the endosteal HSC niche. From Benjamin J. F. et al., Curr Opin Support Palliat Care 2008

Figure 21 .

 21 Figure 21. The hematopoietic stem cell microenvironment Interactions between HSCs and components of their microenvironmental niche regulate stem cell fate. Adapted from: Watt S., Br. J. of Haematology 2001

  contraction of the stem cell pool [171]. Tiu R. et al., have summarized the theories, which can be put forward to explain the initial steps in the evolution of clonal stem cell diseases such as MDS. The defective stem cells may exist in a quiescent form and the abnormal stem cells carrying the genetic defect are more likely to be recruited for proliferation and to initiate the clonal evolution in the context of an overall depletion of the stem cell pool (Figure 22) [171].

Figure 22 .

 22 Figure 22. Mechanism leading to clonal expansion in bone marrow failure Three possible hypotheses to explain evolution of clonal disease are illustrated. A stem cell may gain a lesion that, in the appropriate environment, leads to clonal expansion due to positive selection. Alternatively, under conditions of stem cell depletion, defective stem cells are more likely to be recruited initiating clonal evolution. From: Tiu R. et al., Leukemia 2007

Figure 23 .

 23 Figure 23. The nature of haematopoietic clonality in MDS.

  process of gradual clonal and subclonal selection. Additional defects may provide a growth advantage; facilitate immune escape and apoptosis resistance[171].An increased prevalence of MDS in the elderly population is consistent with the accumulation of abnormal stem cells or deletion / depletion of normal stem cells. The best evidence for the pre-existence of many defective stem or progenitor cells could be provided if cytogenetic analysis is individually performed on purified precursors at the time when haematopoiesis is still polyclonal[171].Another question that arises is which level of haematopoietic ontogenesis is initially affected by the clonal chromosomal defects. In MDS, the answer depends on the type of lesion taken into consideration. Thus, the cytogenetic abnormalities of chromosomes 5, 7, and 20 have been shown to be present in early multipotent CD34 + CD38 -Thy1 + cells[170]. In contrast, trisomy 8 was found only in myeloid lineage-restricted progenitors [170],[171]. In MDS with deletion 20q abnormality apparently the common precursor for myeloid and B-lymphocytes is affected[171].

  Thus, the polymorphisms for two genes: for NAD(P)H: Quinone Oxidoreductase (NQO1), which plays a critical role in detoxifying benzene metabolites[175], [185], [186], and for glutathione S-transferases (GST) are strongly associated with an increased incidence of haematological malignancies [175], [187], [188], [189]. II-3.1 Pathophysiology of MDS Recent research has revealed a further dimension to this pathophysiology, with extrinsic immunological and microenvironmental factors compounding the intrinsic stem cell defect and contributing to the pancytopenia and possibly to leukemic progression.

  Two possible mechanisms could be responsible for the increased rate of apoptosis encountered in the early stages of MDS, i.e., the apoptosis induced by soluble factors, cytokines synthesized by marrow mononuclear cells and mesenchymal stromal cells, and those related by direct cellular contact[175],[197],[201],[202],[203],[204]. In MDS, many studies link over-synthesis of TNF-α by BM MNCs to cell death[175],[202],[205],[206].Furthermore, this cytokine was proved to have an inhibitory effect to both normal and MDS haematopoietic colony growth, indicating that residual normal haematopoiesis can also be blocked in MDS[175],[202],[203].IFN-γ, IL-1, and TGF-β [175], [201], [207] as well as undefined factors produced by stromal cells have also been implicated in causing apoptosis [175], [208], but their role in producing marrow failure has not been well established. The identification of TNF-α as a key cytokine in cell death regulation and the increased susceptibility of MDS cells to TNF-α were the basis of several clinical trials of TNF-α inhibitors, which support their use to improve cytopenia [175], [201].Moreover, the significantly higher levels of the caspase-3 were detected in the BM MNCs selected from MDS patients, which correlate with TNF-α level of cultures supernatant and with increased apoptotic index of haematopoietic precursors[209].These observations were sustained also byArimura K, in 2004, who noticed that matrix metalloproteinase inhibitors (MMPIs) could inhibit the apoptosis priming in MDS BM cells via the inhibition of TNF-α and probably by soluble FasL secretion [210]. Angiogenesis Data showing a significant increase of BM microvascular density (MVD) in MDS compared with normal controls have been reported. Surprisingly, it has been shown that MVD significantly decreases upon transformation to acute leukaemia and that MVD is significantly lower in de novo AML than in MDS [211]. Moreover, the bone marrow microvascular density correlates with significant increases of serum levels of pro-angiogenic cytokines, such as basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF) and tumour necrosis factor-alpha (TNF-α) [208], vascular endothelial growth factor (VEGF) [213], angiogenin, angiopoietin-1, platelet-derived growth factor, EGF(epidermal growth factor), and TGF-alpha and beta (transforming growth factoralpha and transforming growth factor-beta) in MDS patients compared with normal settings [214]. Most of these angiogenic factors appear to be secreted by the neoplastic haematopoietic cells and appear to promote the growth and proliferation of the leukemic cells in an autocrine fashion. More importantly, angiogenic factors play a significant role in the clinical behaviour and outcome of both AML and MDS [214]. VEGF seems to be the best prognostic factor for evaluating the microvascular density, because high levels of this cellular protein were proved to correlate with short survival in MDS patients [215]. Likewise, endothelial cells release increased amounts of VEGF-C, another VEGF family member, in response to leukaemia-derived pro-angiogenic and proinflammatory cytokines such as b-FGF and IL-1, respectively. In turn, interaction of VEGF-C with its receptor VEGFR-3 (FLT-4) promotes the survival and proliferation of a subset of blast cells, and protects them from chemotherapy-induced apoptosis [216]. Immune Dysfunction in MDS Hamblin et al. have raised the possible association between MDS with an autoimmune process, noting the emergence of autoantibody production, and monoclonal lymphocyte proliferation in some patients with MDS [175], [217]. The occasional finding of T cell clonality in MDS has been interpreted as evidence of T cell involvement in the stem cell disorder [175], [218]. MDS also shares some of the features of acquired aplastic anaemia (AA), a disease with well-known autoimmune aetiology [175], [219]. Both in AA and MDS, plasma TNF-α and IFN-γ levels are high and T cell-mediated myelosuppression held [175], [220]. The inhibitory effects of T lymphocytes on autologous granulocyte [175], [221], [222] or erythroid colony growth [175], [223] were also reported. These observations strongly suggest that, as in AA, an autoimmune T cell-mediated myelosuppression contributes to the cytopenia of MDS and that might be considered an immunosuppressive treatment in MDS which could restore the marrow functionality. A Model of Disease Progression in MDS A hypothetical model of MDS disease progression was proposed a decade ago [170], [175]. Transformation of normal stem cells induces danger signals or antigenic changes, hence an autoimmune T cell response directed against the marrow cells. Both MDS clones and normal marrow cells, at various stages of differentiation, are directly inhibited by CD8 + T lymphocytes causing varying degrees of stem cell failure. The persisting autoimmune attack results in chronic overproduction of pro-apoptotic cytokines, especially TNF-α. This synthesis affects committed or mature cells and may contribute to a dysplastic morphology and increased apoptosis in the marrow. Despite the increased cell proliferation in MDS, the marrow fails to export sufficient cells into the blood because intramedullary apoptosis mechanism prevails over proliferation [175].Bone marrow microenvironment as contributor to drug resistanceThe soluble factors produced by bone marrow cells mediate the tumour cells homing, survival and proliferation, and the integrin-mediated adhesion sequesters the tumour cells to the microenvironmental niche[224]. All these processes are involved in the environmentmediated drug resistance. We summarize in the following some mechanisms responsible for the acquired resistance.The BM stromal cells are regarded the main source of chemokine. They constitutively express the stromal cell-derived factor-1 (SDF-1 or CXCL12), implicated in tumour cells homing through the CXCR4 / SDF-1 axis, and in tumour cells survival and adhesion through CXCR7 binding. Moreover, the CXCR4 binding promotes enhances of VLA-4-mediated adhesion to the extracellular matrix components such fibronectin in solid tumours[224].

  chromosome fragments[171]. The major drawback of FISH is the limited number of probes directed towards the suspected targets[171]. The current FISH panels for MDS include probes for detections of -5/del(5q), -7/del(7q), del(20q) and trisomy8 [229].In order to improve the sensitivity of cytogenetic analysis, new karyotyping technologies have been developed including single nucleotide polymorphism array-based karyotyping (SNP-A) and oligonucleotide array-based comparative genomic hybridization array (CGH-A). The major advantages of such technologies are the precise genomic scanning and the fact that they do not require cell division [171]. SNP-A karyotyping is useful in detecting loss of heterozygosity (LOH), even in cases without loss of ploidy, such as uniparental disomy (UPD), in evaluating abnormal clone dimensions relative to the total cell population present in the sample, and facilitating identification of cryptic lesions in bone marrow failure cases with normal or abnormal cytogenetics [171], [229]. Moreover, the SNP arrays may allow the identification of the invariant lesions associated with specific clinical phenotypes [171].Comparative genomic hybridization (CGH) allows detecting changes of chromosome copy number by hybridization techniques between normal and tumour cells [172].The parallel use of a combination of the three cytogenetic technologies -i.e., MC, FISH, and SNP-A -improved the detection rates of genetic abnormalities encountered in patients with MDS (e.g. for del(5q), -7/del(7q), trisomy 8, and del(20q) the detection rate being increased to 35% (MC+FISH), 38% (MC+SNP-A), 38% (FISH+SNP-A) and 39% when all three methods were applied[229].II-4.1 Molecular AbnormalitiesChromosomal aberrations and genetic mutations play a pivotal role in the pathogenesis of MDS and MDS / AML. According to a proposed multistep pathogenesis of leukemia, after the initial damage of the progenitor cell, several additional alterations may affect these cells, providing them with a growth advantage (reviwed inBlau O et al [230]). The most frequent mutations along with their bilogical and clinical significance encountered in these pathologies are summarized in

Figure 24 .

 24 Figure 24. Schematic representation of the multiple-step molecular pathogenesis of RARS-T

Bennett

  et al., refined the system, in 1982, by introducing two supplementary parameters, i.e., the actual monocyte counts in PB and the presence of Auer rods in blasts [172]. To improve prognostic utility, the World Health Organization (WHO) published in 2001 a new standard classification system, which taken in consideration the cytogenetics findings [240]. Thereafter WHO classification has undergone a major revision in 2008, and the most relevant changes are:

III- 1 .

 1 IntroductionFocal adhesions (FAs), named also focal contacts, comprise dynamic groups of structural and regulatory proteins that link transmembrane receptors, such as integrins, to cell cytoskeleton and mediate signals involved in cell attachment, migration, differentiation, proliferation, and gene expression[244],[245].

  Focal adhesions, unlike other structures involved in cell adhesion (gap junctions, tight junctions, desmosome, and hemidesmosome), are not detected as prominent structure under usual electron microscopy [245]. However, immunoelectron microscopy revealed that focal adhesions are dynamic structures which form at the anchorage site of cells to extracellular matrix (ECM), along with adhesion complexes, fibrillar adhesions, and podosomes (Figure 25) [244], [246].

Figure 25 .III- 2 .

 252 Figure 25. Cell adhesion model (A) Overview of cell-to-cell and cell-to-extracellular matrix (ECM) adhesions. Figure adapted from http://www.erin.utoronto.ca (B) Molecular architecture of focal adhesions formed at the anchorage site of cell to ECM. From Mitra S. K., Nature reviews: Molecular Cell Biology 2005

Figure 26 .

 26 Figure 26. Schematic diagrams of FAK structure.From Golubovskaya V.M. and Cance W.G., International Review of Cytology 2007

  overexpressed FAK [252], [274]. FAK expression increases in a stepwise fashion in cell lines derived from various stages of the mouse skin carcinogenesis model [259], [275]. Recent evidence based on the use of an Inducible Cre-LOX model, which deletes FAK expression specifically in epidermal cells and in the hair follicles, has revealed that FAK contributed to both tumour formation, and the acquisition of malignancy [264]. III-4.2 FAK phosphorylation in cancer FAK phosphorylation at different sites can be an imprint of tumour transformation of tissues and could explain some aspects of cancer behaviour [259]. Thus, the phosphorylation of FAK at Tyr397 site has been noticed in invasive tumours, but not in normal epithelium for instance [259], [276]. Likewise, other studies have shown increases of phospho-FAK-Tyr397 in different tumour types, such as carcinomas (cervical [277] and squamous cell carcinoma of larynx [278], as well as in haematological cancers [279], [280]. Moreover, FAK-Tyr397 phosphorylation is followed by the phosphorylation of other catalytic domains, such as Tyr576, Tyr577, Tyr407, Tyr861, Tyr925, which allow the full enzymatic activity of FAK or recruiting other signalling pathways [259]. FAK-Tyr925 phosphorylation was reported in colon cancers and was associated with Ecadherin deregulation during Src-induced epithelial-mesenchymal transition [259], [281]. In addition, FAK-Tyr861 phosphorylation is the imprint of VEGF stimulation and could be involved in tumour vascularisation [259], [269], and [282]. These differences of FAK phosphorylation favour the activation of different signalling pathways downstream (Figure27). Phosphorylation at Tyr397 and Tyr925 causes the increased complex formation between FAK and its SH2-proteins, such as SRC, SHC, p85 (a phosphatidylinositol 3-kinase regulatory subunit), phospholipase Cγ (PLCγ), growth factor receptor bound protein 7 (GRB7), GRB2, p120RHOGAP, which leads to mediate the signalling pathways involved in growth and survival[259]. The Src-specific FAK-Tyr925 phosphorylation is proposed to link FAK to the Ras-MAPK pathway, which is associated with adhesion changes responsible for epithelial-mesenchymal transition[283].

Figure 27 .

 27 Figure 27. FAK phoshorylation regulates downstream signalling pathways. From McLean G.W., Nature reviews: Cancer 2005

Figure 28 .

 28 Figure 28. Model of the possible contributions of FAK in carcinogenesis. From McLean G.W., Nature reviews: Cancer 2005

- 5 .

 5 FAK targeted therapy Two companies have developed ATP-competitive inhibitors to FAK, such as TAC-544 and TAE-226 (Novartis), and PF-228 (Pfizer). TAE-226 inhibits FAK, Pyk2, and the insulin-like growth factor I (IGF-I) receptor, blocks cell proliferation in culture, prevents cell invasion through Matrigel, and increases apoptosis in xenotropic tumour models. Until now, these molecules have proven effective in glioma and ovarian mouse models [265]. Heat shock proteins (HSPs) are a group of proteins, which act as molecular chaperones, binding other proteins in order to ensure their proper folding in the cytosol, endoplasmic reticulum, and mitochondria [293], [294]. They are also involved in intracellular transport of client proteins, in repairing or in proteasome degradation of those partially denatured by exposure to environmental stresses [294], [295], in controlling regulatory proteins, and in refolding of misfolded proteins [294]. IV-2. Heat shock protein 90 structure and functional features related to the conformational structure HSP90 is one of the most abundant cellular chaperone proteins, formed by three structural domains: a highly conserved ATP binding domain near its N-terminus, the middle domain which is the major site of client protein binding, and the C-terminal domain which contains the dimerization interface and a conserved motif responsible for binding TPR-containing cochaperones (Figure 29) [294], [296], [297]. HSP90 differs from other chaperones by the fact that its client proteins are tyrosine kinases (p185 erbB2 [Her-2/neu], FAK, Akt), serine/threonine kinases (Cdk-4 and Raf-1), transcription factors (steroid hormone receptors, mutated p53, HIF-1α, or transcription factors responsible for regulating oncoproteins FLT3, BCR-ABL, EGFR, CRAF, BRAF, MET, VEGFR) [266], [267], [268], [294], [296], [297].

Figure 29 .

 29 Figure 29. Structure of HSP90 dimer. Adapted from Whitesell L. and Lindquist S.L., Nat Rev Cancer 2005

  )[294],[299].In the following, we review some signalling pathways involved in cancer development of which HSP90 is part:-HSP90 controls the Akt kinase activity, involved in cancer progression by its stimulation in cell proliferation and by suppressing apoptosis; -HSP90-BCL-ABL complexes conduce to resistance to traditional chemotherapeutic agents; -HSP90-mutated p53 complexes conduce to disruption of normal transcriptional activity of this protein involved in cell cycle arrest and apoptosis; -HSP90 controls the ubiquitination and proteasome degradation of HIF-1α (a transcription factor that controls the expression of many genes, and thus, of their protein products which play a crucial role in tumour growth, angiogenesis, glucose transport and glycolysis); -HSPs (including HSP90) controls steroid-dependent processes, by their associations in complexes, which are required to maintain the receptor in a conformation capable to binding hormone; -HSP90 through its implication in the turnover of growth factors and other proteins, contributes to resistance to anti-growth signals, to promotion of angiogenesis, tissue invasion and metastasis[297].

Figure 30 .

 30 Figure 30. Model of the possible contributions of HSP90 in carcinogenesis.

  thought to occur due to Src inhibition, ERB-B2 degradation, modulation of oestrogen and progesterone receptors, signalling inhibition of AKT, PI3K, or FLT3 pathways, modulation of angiogenesis, by preferential degradation of mutant BRAF (a client protein of HSP90 which is mutated in most myeloma cases or in colon cancers), or mutant EGFR (such as in lung cancer), by abrogation of ZAP-70 activity and by inhibition of P-glycoprotein function [299].

  groups. The most common isolation methods are based on the ability of MSCs to adhere to plastic or on the use of MSCs surface epitopes, such as specific markers or adhesion molecules.Despite controversies[302], previous work has shown that adherent layers of BM stromal cells from patients with myelodysplasia achieved confluency at a significantly slower rate than those of normal donors [168],[190], [303].Boudard et al. also showed that the stromal layer defects in MDS are associated in some cases with caspase-3 activation and increased angiogenesis[190]. Moreover, there is evidence that these abnormalities affect the functionality of haematopoietic compartment. Thus, in 2001, Tauro et al. showed that the stromal defects in MDS are of heterogeneous origin: altered matrix molecules and changes in superoxide cellular levels, which may contribute to abnormal survival and development of haematopoietic cells [304]. Thereafter, Tauro's team showed that this abnormal function of stromal cells in patients with MDS may contribute to increased apoptosis of haematopoietic cells within the marrow microenvironment and this effect appears to be due to the close HSCto-stromal cells contacts, rather than the release of soluble factors [304]. The haematopoietic deficiencies have been also related to the abnormal expression of soluble factors, such as IL-1β [190], [303], TNFα, and VEGF [190]. Long-term bone marrow cultures on myelodysplastic stroma revealed two broad patterns of haematopoietic progenitor cells (HPC) growth: a first group of cases included the patients whose cultures showed an abnormally low production of HPCs rapidly declining, i.e. within four weeks, to levels outside the normal range. The second group is the one where HPCs proliferation maintained within the normal range for at least seven weeks, albeit at the lower range limit [302]. In Coutinho et al. study, there was a predominance of RA and RSA cases, over other MDS (FAB classified) subtypes, of which the HPC cultures present comparatively normal growth [302]. Regarding the surface antigenic profile of MDS stromal cells vs. normal controls, there are not many references, although most of these antigens are adhesion molecules, having an important role in mediating MSCs-HPCs contacts. Thus, Lopez-Villar et al. noticed a diminution of endoglin (CD105) and integrin β4 chain (CD104) expression on MDS-MSCs [164]. Moreover, the same study indicates that the microenvironmental changes are accompanied by genomic aberrations of MSCs selected from MDS patients [168], [305].

1 ) 2 ; 2 )

 122 the World Health Organization classification of myeloid neoplasms and acute leukaemia; † Hb = haemoglobin; † † PB Mo = peripheral blood monocyte count; ‡ RS = ringed sideroblasts; § Transfusion dependence: + = transfusion-dependent, and -= not transfusion dependent; ¶ Human colony-forming unit: BFU = burst-forming unit-erythroid; CFU-GM = colony-forming unit-granulocyte, macrophage; # RCUD-= Refractory cytopoenia with unilineage dysplasia: -RA = Refractory anaemia; -RT = Refractory thrombocytopoenia; † † † RCMD = Refractory cytopenia with multilineage dysplasia; ٭٭ RARS = Refractory anemia with ringed sideroblasts ; ‡ ‡ RAEB = Refractory anaemia with excess blasts; § § AML inv(3)(q21q26) = Acute myeloid leukaemia with inv(3)(q21q26.2); ♦ too few metaphases; ND=not done; N/A=not applicable Heparinised BM specimens are ficollated with Lymphoprep TM (AXIS-SHIELD PoC AS, Oslo, Norway) by centrifugation at 1500r/min, 30 min, 20 o C, accelerationdeceleration Cells were washed 2 times: first time with RPMI 1640 medium (Eurobio, Les Ulis, France) and second time in Phosphate Buffered Saline (PBS) (Sigma-Aldrich) by centrifugation at 1500r/min, 10 min; 3) Number of cells were counted in Türk camera; 4) 2 -2.5 millions of cells were added by T25 culture flask (25 cm 2 ) in 10 ml MesenCult ® Complete Medium (StemCell Technologies, Vancouver, BC, Canada); 5) Cells were incubated for 3 days at 37 o C in an atmosphere of 95% air and 5% CO2; After 1-2 days of cultures, cells which did not adhere or which are detached progressively are removed by changing medium: ½ new medium and ½ supernatant from 1 st culture (BM stromal cells are isolated from haematopoietic cells due to their capacity to adhere to plastic surfaces); 6) Thereafter, the medium was changed twice weekly and replaced with half-new medium and half supernatant removed by culture until confluency is reached (80% confluency was obtained in 30-35 days). 7) After 80% confluency is reached, cells are harvested. Culture medium was removed and cells were washed once with RPMI 1640 without FCS. MesenCult ® Dissociation Kit (StemCell Technologies) (2-3ml / 25cm 2 flask, warmed in advance) was used to detached cells from culture flasks: cells are incubate in MesenCult®-ACF Enzymatic Dissociation Solution (StemCell Technologies, Catalog #05427) at 37 o C until they were detached (2-3 minutes usually; under microscopic control). The flasks were gently tapped to detaching the remaining cells. 8) 3 mL MesenCult®-ACF Enzyme Inhibition Solution (StemCell Technologies) was added in each flask to inhibit reaction. Cells were collected into a 25 mL conical tube and wash with 5 ml MesenCult Complete Medium (StemCell Technologies)

Figure 31 .

 31 Figure 31. Experimental strategy of STRO-1 / CD73 Immunomagnetic Selection

1: 50 )

 50 , and PE-conjugated mouse anti-human CD73 mAb (1:25), FITC-conjugated mouse antihuman Paxillin monoclonal Ab (mAb) (final dilution 1:50, clone 349/Paxillin, BD Bioscience), PE-conjugated mouse anti-human CD73 mAb (1:25, clone AD2, BD Pharmingen ™ ), unconjugated rabbit anti-phospho-specific FAK [pY 397 ] polyclonal Ab, (1:100, Invitrogen Corporation); mouse anti-human p130CAS mAb (1:40, clone 35B.1A4, Santa Cruz Biotechnology, Inc.), PE-conjugated mouse anti-human HSP90α/β mAb (1:40, clone F-8, Santa Cruz Biotechnology, Inc.), FITC-conjugated mouse anti-human IgG2a,k mAb (1:50, clone G155-178, BD Pharmingen ™ ), PE-conjugated mouse anti-human IgG1,k mAb (1:40, clone MOPC-21, BD Pharmingen ™ ), rabbit anti-human IgG polyclonal Ab (1:100, DakoCytomation). Secondary antibodies (Invitrogen Corporation) were used at following dilutions: FITC-conjugated goat anti-mouse IgG (H+L) 1:50, PE-conjugated goat anti-mouse IgG (H+L) 1:25, Alexa Fluor 633-conjugated goat anti-rabbit IgG (H+L) 1:100 in Ab buffer. Incubation was performed overnight for primary antibodies and for 2 h for secondary antibodies at 4°C, in a moist chamber. Fixation was stopped by washing the cells 3 times with in PBS, pH 7.2.

  Stromal microenvironment is a target of the oncogenesis process as instrument in the control of hypoxia, acidosis and interstitial homeostasis balance, indisputable links of tumor progression. The first article exploited the fundamental information regarding the intercellular dialogue (MSC-MSC and HPC-MSC) in order to define their physiological role in bone marrow microenvironment equilibrium. His reactivity was observed during progression in the MDS risk groups, each of these entities surprising us by behaviors harmonized to haematopoietic partner requirements.

Figure 32 . 1 - 1 .

 3211 Figure 32. Experimental strategy of MSCs selection and functional assays

Figure 33 .

 33 Figure 33. Representative exemples for stromal cells cultures at week 4 in RPMI-1640 A) Normal cells (score confluency 3); B) 50% confluency in RA case (confluency 2); C) no confluence in a RCMD case (score 0) (Magnification 20 x)

  Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'.In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. Thus, was noticed the role of FAK Tyr 397 phosphorylation in promoting cell survival, cell proliferation and cell invasion by FAK-Src signalling pathway, and the recruitment of FAK and paxillin to β1 integrin to promote cancer cell migration via mitogen activated protein kinase activation [265], [317],[318].

Figure 34 .

 34 Figure 34. Experimental strategy of focal adhesion proteins evaluation on CD73 + MSCs Lab-Tek cultures

  highly co-localization to pFAK[Y 397 ] and HSP90αβ in RAEB MSCs, with formation of functional active complexes with nuclear disposition, was correlated with a proliferative advantage in S-MSCs (p<0.01, r=0.79, Spearman test) and less significant in L-MSCs (p<0.05, r=0.64) (Figure35).

Figure 35

 35 Figure 35. Correlations between paxillin expression and the proliferation rates of CD73 + selected MSCs

Figure 36 .

 36 Figure 36. pFAK [Y 379 ] expression in MSC vs. the clonogenic potential of HPCs selected from same cases

  samples were surface-stained with CD45-PE-Cy5 (clone J33, Beckman-Coulter France, Villepinte, France) and CD34-FITC (clone 8G12, BD Biosciences, San José, CA, USA) antibodies for 15 mn at room temperature. Then, the cells were washed and fixed with 3.7% paraformaldehyde for 20 mn. The staining of intracellular proteins was performed after permeabilization in 0.2% Triton X100 (15 mn at room temperature). The specific antibodies for this study were HSP90-phycoerytrin (PE)-conjugated antibody (Clone F8 SCL3-119 which recognizes both HSP90alpha and beta isoforms, Santa Cruz Biotechnology, Santa Cruz, CA), FAK-PE (clone H-1, Santa Cruz), pFAK-PE (clone K73-480, BD Biosciences), pAkt (S473)-AL FL647 (clone M-89-61, BD Biosciences), Akt-AL FL647 (mouse, clone 55PKBa/Akt, BD Biosciences). Cells were incubated for 1 hour at room temperature, washed and re-suspended in PBS before analysis. The isotype controls used for the phospho-proteins

  also show in our model a downregulation of FAK and pFAK after exposure to 17-AAG. FAK is a cytoplasmic protein tyrosine kinase localised to regions called focal adhesions. Many stimuli can induce activation of FAK, including integrins and growth factor. The major site of autophosphorylation, tyrosine 397, is a docking site for the SH2 domains of Src family proteins. Phosphorylated FAK binds and activates proteins forming the FAK complex, and facilitate the generation of downstream signals necessary to regulate cell functions, like motility, survival and proliferation. Dysregulation of FAK could participate in the development of cancer, and abnormal activation of FAK has been described in AML. Our data suggest that FAK, as a client of HSP90, could be indirectly targeted by HSP90 inhibition.

  Figure 1

  line with previous data,[168],[190],[303], we noticed the confluence deficiencies and spontaneous lysis in 25-50% of MDS stromal cells amplified during 30-35 days in vitro cultures, especially in refractory cytopenia (RC) cases. Moreover, stand out also the different distribution of the three morphotypes of MSCs (rounded-shaped; thin, spindle-shaped; and large, flat cells) in MDS layers compared to normal settings. The slight decrease in the number of large, flat cells in refractory cytopenia with unilineage dysplasia (RCUD) settings, which is considered the onset of terminal differentiation, in addition to the size differences within this population among the different study groups are worth noting. These characteristics could indicate maturation defects.

  At the cellular level, MDS stromal cells depict dysplastic changes very similar those met in haematopoietic compartment, and issues related to altered actin organization, such as thin and flat morphologically altered cells, as Ilić et al. have already reported in mouse FAK (-/-) fibroblasts [256]. In addition, we have compelling arguments that MSC from RCUD settings show a continuous decline of proliferation and a reduced clonogenic capacity during 14 days of CFU-F cultures, in the absence of signals from hematopoietic cells, and these growth deficiencies are significantly correlated with diminution of CD44 and CD49e (α5-integrin) adhesion molecules. Besides these obvious, the last decade provides evidence about the cytogenetic abnormalities of MSCs selected from MDS patients [168], [230], and [305]. Of note is the fact that the MSC cytogenetic aberrations differs from the haematopoietic cells chromosomal abnormalities of the same subjects [230]. Moreover, the Blau et al. study revealed that only a fraction of the culture cells harbor cytogenetic alteration, that support the hypothesis of abnormal MSC clones existence, which probably are favored during malignancy advancement. In addition, Varga et al. proves a CAFC (cobblestone area-forming cells) frecvencies diminution when normal hematopoietic cells were incubated on MDS stromal cells, compared to normal stromal layer-containing control cultures [322]. Furthermore, the same team noticed a marked reduction in the plasticity of mesenchymal stem cells of MDS patients compared with those of normal marrow donors, in neurogenic and adipogenic differentiation ability and hematopoiesis supporting capacity in vitro [322].

  The first description of the in vitro MSC niche belongs to Prockop DJ which argues the different roles fulfilled by the cellular subpopulations within this niche. He noticed in stationary MSC cultures the colonies comprising two major regions, the inner region populated by the more commitment precursors, and the outer one formed by a population of cells that express surface proteins with an inhibitory influence on cell adhesion [such as α 6integrin and podocalyxin-like protein (PODXL)]. The last ones are highly motile, secrete DKK-1 (an inhibitor of the canonical Wnt signalling pathway), and serve as nurse cells for other subpopulations; thus, they are key elements of the rapid growth phase[134].

Figure 37 .

 37 Figure 37. Model of FAK signalling pathways possibly involved in intrinsic proliferation of MSCs

Figure 38 .

 38 Figure 38. Model of the possible contributions of FAK expression to the influence of MSCs on the clonogenicity of haematopoietic precursors

4 .

 4 Our preliminary data justify the rising interest concerning the untapped potential of microenvironmental prognostic factors as distictive item of MDS lesion, and sustain the further validation, in a broader context, of the diagnostic value of microenvironmental entities inventory as part of phisiopathological process evaluation in MDS. 5. The perception of the stroma-related disease mechanisms may underlie to the development of alternative therapeutic approaches.In this article, we bring compelling evidence that BM MSC from both, AML patients and healthy subjects, conferred a substantial beneficial effect on AML cells throughout the HPC-BM MSC co-culture system. Herein, we investigated the influence of BM MSCs on in vitro modulation of cytokine secretion (IL-1b, TNF-a, IL-10, IFN-g, IL-4, IL-5, and IL-2) by AML cells, as well as, the supportive capacity of soluble factors synthesized by BM MSCs on AML cells. Significant differences were observed between AML-BM MSC and normal-BM MSC with respect to IL1b secretion that was upregulated in AML cell cultures, both after 24 and 72 hours.

Table 1 Citations in PubMed as of 17 August 2011 Table 2 Phenotypic characterization and expression of growth factors of human MSCs Table 3 Inductive factors for in vitro mesenchymal lineage differentiation Table 4 Myelodysplatic Syndromes pathophysiology Table 5 Invariant balanced and unbalanced chromosomal abnormalities detected by metaphase karyotyping and FISH Table 6. Recurrent gene mutations in MDS and MDS/AML settings Table 7 Peripheral blood and bone marrow findings in MDS (The 2008 revision of WHO classification) Table 8 IPSS Prognostic Score System Table 9 Focal adhesion components Table 10 Clinical and laboratory characteristics of MDS patients and control subjects studied Table 11 Antibody clones, isotype and source x BIBLIOGRAPHICAL PART Chapter I -Preliminary Basis: The Bone Marrow Haematopoietic Niche

 1

Table 1 Citations in PubMed as of 17 August 2011

 1

	Search name	Citations
	CFU-F (colony-forming unit fibroblastic)	457
	Marrow stromal cells	10042
	Mesenchymal stem cells	15073
	Mesenchymal stromal cells	4107
	MSCs	5608

Adapted from: Prockop D. J., Molecular Therapy 2009

A position statement of the International Society for Cytotherapy proposed to use the term "multipotent mesenchymal stromal cells"

[106]

.

Table 2

 2

summarize the other phenotypic markers and growth factors expressed by MSCs amplified in culture [129].

Table 2 Phenotypic characterization and expression of growth factors of human MSCs

 2

	Common name	CD designation	Detection
	Adhesion molecules*
	ALCAM	CD166	Pos
	ICAM-1	CD54	Pos
	ICAM-2	CD102	Pos
	ICAM-3	CD50	Pos
	E-selectin	CD62E	Neg
	L-selectin	CD62L	Pos
	P-selectin	CD62P	Neg
	LFA-3	CD58	Pos
	Cadherin 5	CD144	Neg
	PECAM-1	CD31	Neg
	NCAM	CD56	Pos
	HCAM	CD44	Pos
	VCAM	CD106	Pos
	Hyaluronate receptor	CD44	Pos
	Growth factors and cytokine receptors*
	IL-1R (α and β)	CD121a,b	Pos
	IL-2R	CD25	Neg
	IL-3R	CD123	Pos
	IL-4R	CD124	Pos
	IL-6R	CD126	Pos
	IL-7R	CD127	Pos
	Interferon γR	CDw119	Pos
	TNF-α-1R	CD120a	Pos
	TNF-α-2R	CD120b	Pos
	FGFR		Pos
	PDGFR	CD140a	Pos
	Transferrin receptor	CD71	Pos
	Integrins*
	VLA-α1	CD48a	Pos
	VLA-α2	CD49b	Pos
	VLA-α3	CD49c	Pos
	VLA-α4	CD49d	Neg	Expressed in long term bone marrow cellule without induction
	VLA-α5	CD49e	Pos	IL-6
	VLA-α6	CD49f	Pos	IL-7
	VLA-β chain	CD29	Pos	IL-8
	β4 integrin	CD104	Pos	IL-11
	LFA-1 α chain	CD11a	Neg	IL-12
	LFA-1 β chain	CD18	Neg	IL-14
	Vitronectin R α chain	CD51	Neg	IL-15
	Vitronectin R β chain	CD61	Pos	LIF
	CR4 α chain	CD11c	Neg	M-CSF
	Mac1	CD11b	Neg	Flt-3 ligand
	Additional markers*			SCF
	T6	CD1a	Neg	Induced by IL-1
	CD3 complex	CD3	Neg	IL-1
	T4, T8	CD4, CD8	Neg	IL-1β
	Tetraspan	CD9	Pos	IL-6
	LPS receptor	CD14	Neg	IL-8
	LewisX	CD15	Neg	IL-11
	-	CD34	Neg	IL-11
	Leukocyte common antigen	CD45	Neg	G-CSF
	5' terminal nucleotidase	CD73	Pos	GM-CSF
	B7-1	CD80	Neg	LIF
	HB-15	CD83	Neg	Not expressed
	B7-2	CD86	Neg	IL-2
	Thy-1	CD90	Pos	IL-3
	Endoglin	CD105	Pos	IL-4
	MUC18	CD146	Pos	IL-10
	BST-1	CD157	Pos	IL-13

Table 3 Inductive factors for in vitro mesenchymal lineage differentiation

 3

	Lineage	Inductive factors
	Adipocyte	dexamethasone, indomethacin, insulin, methylisobutylxanthine, thiazolidinedione
	Chondrocyte	ascorbate, bone morphogenetic protein 6, dexamethasone, transforming growth
		factor β
	Endothelial	ascorbate, epidermal growth factor, fibroblast growth factor 2, vascular endothelial
		growth factor
	Hematopoietic	co-culture models
	support
	Muscle (cardiac)	5-azacytadine
	Muscle (skeletal) low serum concentration, horse serum
	Muscle (smooth)
	Osteoblast	ascorbate, bone morphogenetic protein, dexamethasone,
		1,25 dihydroxy vitamin D3
	Tendon	mechanical stimuli

Adapted from: Gimble J. M., Transfusion Medicine and Hemotherapy 2008

  /glycophorin A -population has a substantial clonogenic capacity (approximately 100-fold, enriched in colony-forming unit-fibroblast [CFU-F]). These cells are capable of generating adherent cell layers containing multiple cell types, including adipocytes, smooth muscle cells, and fibroblastic elements; furthermore, this population displays a greater ability to maintain the normal development of the human myeloid lineage than the stromal cells that are commonly isolated from unmanipulated BMs[123]. More recently,Gronthos et al.

	provided evidence that osteogenic precursors are present in the STRO-1 + fraction of human
	BM cells [148]. Psaltis et al. also found a strong correlation between the amount of STRO-1
	with mRNA expression of transcription factors related to cellular proliferation and
	differentiation, which have been associated with an immature, stem-like phenotype [149].
	CD73 expression has also been observed in different cells, and its physiological role is to

, [147]. Plasma membranebound ecto-5'-5'nucleotidase (CD73) has been proposed as the most useful molecule for developing robust in vitro MSC assays [147]. However, Simmons et al. reported that the STRO-1 + metabolize adenosine 5'-monophosphate (AMP) to adenosine [150]. CD73 acts as a signaltransducing molecule in the human immune system (specifically, it acts as a co-stimulatory molecule in T cell activation), and it has been shown to be involved in controlling lymphocyte-endothelial cells interactions [151]. It has been hypothesized that CD73

II-1. The Myelodysplastic Syndromes: Introduction

	The myelodysplastic syndromes (MDS) represent a heterogeneous group of myeloid
	neoplasms characterized by abnormal differentiation and maturation of myeloid cells, bone
	marrow failure, manifested by peripheral blood cytopenias and hypercellular bone marrow,
	although in one quarter of the cases the bone marrow could be hypocellular, and a genetic
	instability with enhanced risk to transform to acute myeloid leukaemia (AML) [162], [163].
	The incidence of MDS disease is estimated of 4 / 100,000 per year, but rises to 30 / 100,000
	for people over 70 years [164]. In children, the annual incidence is 1-2 / million [165]. The

median age decreased dramatically in recent years being 51 years in a recently published study, and the distribution of MDS subtypes demonstrated a markedly low incidence of MDS with deletion 5q (0.9%)

[166]

. In terms of gender, distribution is slowly greater in males

[164]

.

Evolution to AML is about 20% and median survival is around

28 months [167]

.

Table 4

 4

	summarizes the pathophysiology of MDS in relation to environmental factors
	exposures.

Table 4 Myelodysplatic Syndromes pathophysiology Theories of Pathophysiology involved in MDS Development Potential targets/Component Involved Overall Result of Abnormality

 4

	Environmental/Aging
	Aging	Increased BM apoptosis

Adapted from: Warlick E.D. and Smith B.D., Current Cancer Drug Targets 2007

Table 6

 6

[175], [231], [232], [233], [234], [235], and [236].

Table 6 . Recurrent gene mutations in MDS and MDS/AML settings

 6

	Genetic	Gene	CRS	Cells affected	Biologic and clinical	Outcomes
	mechanism			by mutation	significance	at diagnosis
	Haploinsufficiency	RPS14	5q33	CD34 + cells	Abnormal erythroid apoptosis [233] differentiation and	Unknown [233]
		miR-145 and miR-146	5q33 CDR	NA	Thrombocytosis [233]	Unknown [233]
		Egr1	5q31	NA	Enhances stem-cell self-renewal [233]	Unknown [233]
		HSPA9	5q31.2	CD34 + cells	Delayed erythroid apoptosis [233] maturation and increased	Unknown [233]
	Mutations in	DNMT3A	2p	HSC/
	regulators of DNA			Unfractiona-
	methylation			ted BM
				samples

Table 7 )

 7 [241],[242].

Table 7 . Peripheral blood and bone marrow findings in MDS (The 2008 revision of WHO classification)

 7

	Entity				Blood findings	BM findings
	Refractory unilineage dysplasia (RCUD): (-cytopenia with	Unicytopenia bicytopenia a	or	Unilineage dysplasia: ≥ 10% of the cells in one myeloid
	RA refractory anaemia; -RN refractory neutropenia; -RT	No or rare blasts (< 1%) b	lineage, < 5% blasts,
	refractory thrombocytopenia)		< 15% of erythroid precursors are ring
						sideroblasts
	Refractory anaemia with ring	Anaemia	≥15% of erythroid precursors are ring
	sideroblasts (RARS)			No blasts	sideroblasts,
						Erythroid dysplasia only,
						< 5% blasts
	Refractory	anaemia	with	Cytopenia(s)	Dysplasia in ≥ 10% of the cells in ≥ 2
	multilineage dysplasia (RCMD)	No or rare blasts (< 1%) b	myeloid lineages (neutrophil and/or erythroid precursors and/or
					No Auer rods < 1 x 10 9 /L monocytes	megakaryocytes), < 5% blasts in marrow,
						No Auer rods,
						± 15% ring sideroblasts
	Refractory anaemia with excess blasts-1 (RAEB-1)	Cytopenia(s) < 5% blasts b	Unilineage or multilineage dysplasia 5-9% blasts
					No Auer rods < 1 x 10 9 /L monocytes	No Auer rods
	Refractory anaemia with excess	Cytopenia(s)	Unilineage or multilineage dysplasia
	blasts-2 (RAEB-2)			5-19% blasts	10-19% blasts
					Auer rods ± < 1 x 10 9 /L monocytes	Auer rods ±
	Myelodysplastic	syndrome-	Cytopenia(s)	Unequivocal dysplasia in < 10% of cells
	unclassified (MDS-U)			< 1% blasts	in one or more myeloid lineages when
						accompanied	by	a	cytogenetic
						abnormality considered as presumptive
						evidence for a diagnosis of MDS (listed
						in Table 6)
	MDS associated with isolated	Anaemia	Normal to increased megakaryocytes
	del(5q)				Unusually normal or	with hypolobulated nuclei
					increased platelet count	< 5% blasts
					No or rare blasts (<	Isolated del(5q) cytogenetic abnormality
					1%)	No Auer rods
	a Bicytopenia may occasionally be observed. Cases with pancytopenia should be classified as MDS-U. b The cases with marrow myeloblast percentage <5%, but a mean of 2% to 4%
	myeloblasts in the blood, should be included in RAEB-1. Cases of RCUD and RCMD with 1% myeloblasts in the blood should be classified as MDS-U. c Cases with Auer rods and <5%
	myeloblasts in the blood and less than 10% in the marrow should be classified as RAEB-2.
	Although the finding of 5% to 19% blasts in the blood is, in itself, diagnostic of RAEB-2,
	cases of RAEB-2 may have <5% blasts in the blood if they present Auer rods or 10% to 19%
	blasts in the marrow or both. Similarly, cases of RAEB-2 may have <10% blasts in the
	marrow, but may be diagnosed using the other two findings: Auer rods and / or 5-19% blasts
	in the blood.

Adapted from Vardiman J. W. et al., Blood 2009

Table 8 . IPSS Prognostic Score System Prognostic variable Score value

 8

			Low	Intermediate Int-1 Int-2	High
		0	0.5	1.0	1.5	2.0
	BM blasts (%) Cytogenetics * Cytopenias **	<5 Good 0/1	5-10 Intermediate 2/3	-Poor -	11-20 --	21-30 --

* good prognosis

: normal, 5q-, 20q-, -Y; poor prognosis: complex aberrations, -7,7q-;

intermediate prognosis: all others, ** haemoglobin <10g/dl, trombocytes <100x10 9 /l, absolute neutrophil count <1.5x10 9 /l. Adapted from: Haferlach T.

and Kern W., Springer Berlin Heidelberg 2006

Table 9

 9

	) [244], [246].

Table 9 . Focal adhesion components

 9

	Location		Focal adhesion proteins
	Extracellular		Collagen, fibronectin, heparan sulfate, laminin, proteoglycan,
			vitronectin
	Transmembrane	Integrins 18α and 8β (24 combinations in humans), LAR-PTP
			receptor, layilin, syndecan-4
			Actin, α-actinin, EAST, ezrin, filamin, fimbrin, kindling, lasp-
		Structural	1, LIM nebulette, MENA, meosin, nexilin, paladin, parvin, profilin, ponsin, radixin, talin, tensin,
			tenuin, VASP, vinculin, vinexin
			Protein tyrosine kinase: Abl, Csk, FAK, Pyk2, Src
			Protein serine/threonine kinase: ILK, PAK, PKC,
	Cytoplasmic	Enzymatic	Protein phosphatase: SHP-2, PTP-1B, ILKAP Modulators of small GTPase: ASAP1, DLC-1, Graf,
			PKL, PSGAP, RC-GAP72
			Others: calpain II, PI3-K, PLCγ
			p130Cas, caveolin-1, Crk, CRP, cten, DOCK180, DRAL,
		Adapters	FRNK, Grb 7, Hic-5, LIP.1, LPP, Mig-2, migfilin, paxillin,
			PINCH, syndesmos, syntenin, Trip 6, zyxin

Table 10 Clinical and laboratory characteristics of MDS patients and control subjects studied

 10

	CFU-GM ¶		296	13	184	180	11	88		72	29	92	256	ND	248	376
	BFU-E ¶		248	8	200	160	12	0		28	19	174	220	ND	240	268
	Transfusion	Dependence §	-	-	-	-	+	-		+	-	-	N/A	N/A	N/A	N/A
	Cytogenetics		46XX	♦	♦	46.XY.del(20)(q11q13)[8]	46.XY	46.XX.del(5)(q22q34)	del(20)(q11q13)[19]	46XX	46XX	46.XY.inv(3)(q21q26)[20]	ND	ND	ND	ND
	RS ‡		-	(+)≥15%	-	(+)<15%	-	(+)<15%		-	-	-	-	-	-	-
	Dys-	megakariopoiesis	-	-	-	++	-	-		-	++	+++	-	-	-	-
	Dys-	erythropoiesis	-	++	-	++	-	+++		-	-	++	-	-	-	-
	Dys-	myelopoiesis	-	-	++	+++	++	+++		+++	+++	++	-	-	-	-
	BM	cellularity	++	+++	++	+++	++	+++		+++	++	+++	++	++	+++	++
	PB Mo † †	x10(9)/L	0.34	0.97	0.41	0.44	0.26	0.21		0.15	0.5	0.7	0.32	0.28	0.59	0.335
	Platelets	x10(9)/L	222	298	84	100	308	324		445	201	254	275	175	389	375
	Leucocytes	x10(9)/L	6.62	9.6	5.81	3.37	3.7	3.21		3.98	2.51	43.34	7.65	6.97	4.69	8.89
	Hb †	g/dL	10.5	12.7	12.2	10.1	9.8	11		9.5	12.7	9.8	14.7	13.5	11.7	15.2
	% blasts	in BM	3.5	1	1	3	2.5	11		7	16.5	34	0	0	1.5	0
	% blasts	in PB	0

Table 11 Antibody clones, isotype and source

 11

	Name	Source	Clone	Conjugate	Isotype
	MSCs specific markers
	anti-STRO-1	Santa Cruz	sc-	FITC	Mouse
	anti-CD73	Biotechnology BD Pharmingen TM	47733 AD2	PE	IgM Mouse
					IgG1
	Endothelium-related markers anti-CD31 BD Pharmingen TM	M89D3 Alexa Fluor® 488	Mouse
	anti-CD106	BD Pharmingen TM	51-10C9 FITC	IgG2a Mouse
					IgG1
	Hematopoietic-related markers anti-CD45 BD Pharmingen TM	2D1	PerCP	Mouse
	anti-CD16	BD Pharmingen TM	B73.1	PE-Cy TM 7	IgG1 Mouse
					IgG1
	Adhesion markers anti-CD29	BD Pharmingen TM	MAR4	PE-Cy TM 5	Mouse
	anti-CD54	BD Pharmingen TM	HA58	APC	IgG1 Mouse
	anti-CD44	BD Pharmingen TM	515	PE	IgG1 Mouse
	anti-CD49e	BD Pharmingen TM	IIA1	PE	IgG1 Mouse
	anti-CD144	BD Pharmingen TM	55-7H1 PE	IgG1 Mouse
					IgG1
	II-5

. Cell Selection II-5.1. EasySep Immunomagnetic Selection Procedure

III.2-1. Focal adhesion proteins evaluation in MSCs selected from MDS settings vs. normal bone marrows

  Multicolour fluorescent microscopy has proven to be a successful technique for FA protein analysis. It allowed us to assess the intracellular localization of proteins, and to understand the spatial relationship between these proteins via colocalization analysis.

	A first remark was the bright nuclear expression of pFAK [Y 397 ] in pathological MSCs, but
	not in focal adhesion contacts as we had expected. Over 80% of RAEB MSCs showed nuclear
	expression of pFAK [Y 397 ].
	For HSP90αβ expression, an increased level, directly correlated with pFAK [Y 397 ] up-
	regulation was found in MSCs cells from RAEB group (2.62-fold in S-MSCs and 3.82-fold in
	L-MSCs). HSP90αβ immunostaining of CD73 + MSCs cultures show that this chaperone is
	abundantly expressed in the cytoplasm, but a nuclear immunoreactivity was also noticed.
	About 30% of normal MSCs also showed an HSP90αβ nuclear expression.
	Difference from normal for RAEB MSCs consists in an overall greater expression of
	cytoplasmic staining and increased percentages of nuclear HSP90αβ positive cells (around
	80%).

Table 1

 1

	: patient characteristics
	_____________________________________________________
	Median age (range)	66 years (11-91)
	Sex (M/F)	99/78
	WHO classification (N)
	RA	20
	RAS	1
	RCMD	24
	RCMD-RS	2
	RAEB-I	38
	RARB-II	55
	5q-	14
	CMML (N)	23
	Cytogenetic prognostic groups (N=152)
	Good	91
	Intermediate	32
	Poor	29
	IPSS (N=152)
	Low	56
	Intermediate-1	44
	Intermediate-2	32
	High	20
	Median follow-up (days)	593
	Median time to transformation (days)	386
	(87 patients)

Table 2 :

 2 Expression of HSP90, pAKT, FAK and pFAK according to IPSS risk category

			Mean Fluorescence Intensity Ratio
		__________________________________________
	IPSS	HSP90	pAKT	FAK	pFAK
		__________________________________________
	Low	54	6	16	25
	Int-1	73	9	21	29
	Int-2	85	13	29	47
	High	86	15	34	51

ACKNOWLEDGEMENTS

Firstly, I would like to express my gratitude to the jury members who made me the honour to judging this work: I thank Prof. Dr. Marc Berger and Prof. Dr. Victor Cristea for their agreement to review my work as rapporteurs and to Prof. Dr. Denis Guyotat and Prof. Dr. Minodora Dobreanu as examiners.

Thereafter I would like to thank my supervisor, Prof. Dr Lydia Campos Guyotat, for giving

(RS-MSCs) Slowly replicating cells (SR-MSCs) Periodic acid Schiff (PAS) Doubling time (DT) Very small embryonic-like (VSEL) stem cells Parathyroid-related peptide receptor (PTH1R) Protein kinase A (PKA) Angiopoietin 1 (ANG-1) Vascular cell adhesion molecule 1 (VCAM 1)

II-4. Cytogenetic and Molecular abnormalities

II-4.2 Cytogenetic Abnormalities

A typical feature of MDS is the presence of invariant chromosomal abnormalities, which, with some exceptions (e.g., chronic myelomonocytic leukaemia [CMML]), atypical chronic myeloid leukaemia (aCML) are mostly unbalanced [171]. The clonal chromosomal defects affect certain chromosomes more frequently (Table 5). The resulting loss of heterozygosity (LOH) or duplications are related to malignant transformation [171]. An alternative solution for the poor proliferation of dysplastic cells, which could be responsible for the low sensitivity of this method, is represented by interphase fluorescent in situ hybridization (FISH), as it does not require cell division, and consequently, cell cultures, being more sensitive than traditional metaphase cytogenetics, allow detection of

EXPERIMENTAL PART EXPERIMENTAL RESULTS

Heat-Shock-Protein (HSP)-90 is overexpressed in high-risk myelodysplastic syndromes

and associated with higher expression and activation of Focal Adhesion Kinase (FAK)

Françoise Solly 1 , Pascale Flandrin The levels of all proteins studied were significantly higher in MNC from patients with refractory anaemia with excess of blasts (RAEB) than in MNC from patients with refractory anaemia (RA) or chronic myelomonocytic leukemia (CMML). The same difference was observed in CD34+ cells. High levels of HSP90, FAK, pFAK and pAKT were associated with shorter survival and increased risk of progression to acute leukaemia.

The effects of inhibition of HSP90 were evaluated in 25 RAEB samples by incubating cells with 17-AAG, an inhibitor of HSP90. A downregulation of HSP90, pFAK and pAKT was observed in MNC and CD34+ cells at 12 hours, associated with increased apoptosis as assessed by activated caspase 3 and annexin V expression.

Our data suggest the implication of HSP90 in the pathogenesis of MDS with excess of blasts and evolution to overt leukaemia, in association with FAK and Akt activation. Moreover this signalling network could be a therapeutic target through HSP90 inhibition.

Survival duration and time to transformation into overt leukemia were significantly shorter in patients with high expression of HSP90, pAKT, pFAK and FAK (fig 3). For all those intracellular proteins, the percent of positive cells were significantly higher in the groups of increasingly poor prognosis as defined by the IPSS (Table 2). In multivariate analysis, we studied the effects of known parameters such as age, cytogenetics (or IPSS), percentage of blasts and marker expression. Only cytogenetics (or IPSS), percentage of blasts and percent of CD34+ cells remained independent prognostic factors.

Transformation into AML

Transformation occurred in 87 cases in total, after a mean delay of 386 days. Nineteen cases could be reevaluated at the time of transformation. This included 9 cases with a diagnosis of RA and 10 RAEB. The blast percentage was 6.5% (±5.9) at diagnosis and 42% (±16) after transformation.

As presented in Figure 4, the levels of HSP90, FAK, pFAK and pAKT were significantly higher after transformation than at diagnosis. This was the case in MNC (p<10 -4 ) and more interestingly in CD34+ cells (p<10 -4 ).

Inhibition of HSP90 (Figure 5)

The effects of inhibition of HSP90 were studied by exposing MNC to 17-AAG for up to 24 hours. Thirty-nine cases of RAEB expressing high levels of HSP90 were studied. In the presence of 2 µM 17-AAG, the percentages of viable cells at 12 and 24 hours were respectively 60 and 32 of control without 17-AAG. At a concentration of 5 µM, the percentage of viable cells was 38% at 12 hours, and no viable cell could be recovered at 24 hours. 17-AAG significantly increased the percentage of apoptotic cells, as assessed by activated caspase-3 expression and annexin V binding. After 12 hours of exposure to 17-AAG 5 µM, more than 90% cells were apoptotic. In clonogeneic assay, the yield of colonies was heterogeneous in the absence of 17 AAG (mean number ± SD of CFU-GM, clusters and BFU-E : 60 ± 80, 139 ± 136, 19 ± 14 respectively. At a concentrartion of 5 µM, 17-AAG completely inhibited the growth of all types of colonies in semi-solid medium.

The effects of 17-AAG on protein expression was studied after 12 hours of culture. The same staining technique was used as for uncultured cells. In addition, we also assessed the expression of HSP70, , which has been reported to be upregulated in the presence of HSP90

inhibitors. The levels of HSP90, FAK, pFAK and pAKT were significantly in the presence of 2 and 5 µM of 17-AAG. This effect was more evident in CD34+ cells than in MNC. By contrast, expression of HSP70 was not significantly modified in both cell types. Thereafter, a low level of CD45 expression in STRO-1 + cells cannot be excluded. A possible explanation for this expression may be the fact that, in non-haematopoietic cells, CD45 functions as a negative modulator of growth factor receptor tyrosine kinases in addition to its well-established role as activator of src family tyrosine kinases [126].

Unlike the STRO-1 positive cells, the STRO-1 -CD73 + were negative for all these markers and displayed increased levels of adhesion markers. This difference in the expression of adhesion markers may reflect the different roles of these cells within their own in vitro niche.

Technically, the two fractions could be exploited differently, STRO-1 + cells being more robust for carrying out in vitro MSC growth assays, whereas CD73 + cells have proven their utility in the evaluation of adhesion profiles. Moreover, PJ Simmons and B Torok-Storb have claimed that a STRO-1 + stroma layer represents a good alternative for an in vivo stroma for performing assays to evaluate HPC-MSC contacts. The STRO-1 molecule does not affect the proliferative abilities of HPCs by itself and it has low affinity for complement. Thus, the STRO-1 layer appears to only provide signals when induced by the engagement of other adhesion molecules [123].

Afterwards, the immunomagnetic selection allowed us to isolate the two major subpopulations that correspond to those previously described in terms of size and growth abilities.

The first population, STRO-1 + CD73 -, was numerically lower in normal BM controls and displayed a rounded shape or long cytoplasmic extensions; in terms of size, cells in this population ranged between 5 and 26 µm. In cytometry, the STRO-1 + cells fell to near 50 on the FSC. These cells met the criteria for a quiescent, slowly cycling population supported by the morphological appearance of resting cells, approximately 2-fold higher clonogenic capacity upon plating and 2.3-fold higher proliferation efficiency at 14 days of culture compared with the STRO-1 -CD73 + fraction.

On the other hand, the STRO-1 -CD73 + fraction included mostly larger (50 to 110 µm, on average) and more granular cells with lower proliferative and clonogenic potential.

What would be the phenotypic features of MDS cultures?

Under the MDS condition, we noticed a higher number of STRO-1 + cells that coexpressed CD106 and CD31 between 20 and 30 days of culture and which persisted until 60 days in the RC group. From the expression of these markers in relation to MDS pathophysiology, two hypotheses can be evoked: the former is related to CD106 upregulation

The diminution of adhesion markers expression on MSC surface interfere with HSC clonogenicity

The preliminary results indicate the fact that the clonogenic potential of HPC is controlled by adhesion mechanisms dependent on stroma, and α5-integrin is one of the molecules involved in this process. This is supported by statistical data which proved that the diminution of α5integrin expression in MSC correlate significantly with the decreased clonogenic potential of myeloid and erythroid precursors, selected from the same cases, and from both groups, RC and RAEB.

In light of these observations, then we intended to decode the focal adhesion (FA) signalling pathways and to understand whether adhesion-mediated processes contribute to transduction of intrinsic proliferative signals, as well as their impact on HPC-to-MSC interactions.

Molecular mechanisms whereby the FA proteins can contribute to the increased proliferation of MSCs selected from RAEB settings

The proliferation differences occur in RAEB cultures compared to normal settings can be attributed both to smaller cells (S-MSCs) as well as to large, more mature ones (L-MSCs), which present the qualitative defects of FA proteins (focal adhesion kinase [FAK], and paxillin), such as intensity differences, nuclear localization, and their association in complexes. Moreover, the MSCs from RAEB cultures highlight a strong complexation of FA proteins to HSP90 in nuclear area, which support a proliferative behaviour of these cells. This hypothesis is confirmed by the experiments in which the pharmacologic inhibition of HSP90 on cells leads to decreased FAK signalling, and consequently, displays growth-inhibitory effects similar to FAK inhibition alone [266]. In addition, this high co-localisation to HSP90 indicates the cessation of proteasome-mediated recycling of these proteins.

There are some possible molecular mechanisms whereby the pFAK [Y 397 ] / HSP90αβ and paxillin co-localization could contribute to the increased rate of proliferation of MSCs (Figure 37).

PERSPECTIVES

Several studies remain to complete these results:

The differential expression of the phenotypic markers, STRO-1 and CD73, could be used for establishing of a cellular hierarchy in the MSC niche?

In order to establish a putative hierarchy of these subpopulations of MSCs (STRO-1 + CD73 - and STRO-1 -CD73 + ), it would be necessary the assessement of the expression patterns of genes, such as Oct-4 (characteristic of embryonic cells), and DKK-1 (an inhibitor of the canonical Wnt signaling pathway). Moreover, the expression of other surface epitopes such as the "anti-cell adhesion proteins," e.g., α 6 -integrin and podocalyxin-like protein (PODXL), can be used for this purpose, as recently suggested by D J Prockop [134].

The distribution changes of MSC morphotypes in primary culture layers could be accompanied by the changes of differentiation potential responsible for the extinction / restriction of a mature cell type?

The differentiation capacities of the MSC subpopulations selected from MDS settings compared to normal controls could raise new questions about the pathogenesis of these diseases.

How integrates our results in the hierarchy of molecular events responsible for the common lesion of HSC and MSC?

To clarify whether these dysfunctions are the result of transdifferentiation abnormalities or dysfunction of a common precursor cells, further studies should address to the cytogenetic characterization and transcriptome decryption of MDS MSCs.

Is α 5 integrin involved in HPC division symmetry?

To confirm the role of α 5 -integrins in the regulation of HPC fate (i.e., clonogenic potential and self-renewing divisions capacity), an in vitro surrogate model for haematopoietic niche could be imagined using the co-cultures of MSCs fractions with CD34 + CD133 + cells, in the absence In this brief communication, we present a case report of a congenital acute leukemia at a child. This entity is a challenge in terms of diagnosis, disease monitoring, and therapeutic decisions due of scarcity of the literature data, and due their unpredictable evolution, occasionally even to spontaneous remission.

In this case, we noticed a blast population without lineage assignment, expressing CD123 (hIL-3-R), normal karyotype, and displaying a resistant phenotype at conventional chemotherapy and to the gemtuzumab ozogamicin therapy. The allogeneic cord blood cell transplantation allowed the achievement of cytological and immunocytometric complete remission.