Involvement of GPx-3 in the Reciprocal Control of Redox Metabolism in the Leukemic Niche.

The bone marrow (BM) microenvironment plays a crucial role in the development and progression of leukemia (AML). Intracellular reactive oxygen species (ROS) are involved in the regulation of the biology of leukemia-initiating cells, where the antioxidant enzyme GPx-3 could be involved as a determinant of cellular self-renewal. Little is known however about the role of the microenvironment in the control of the oxidative metabolism of AML cells. In the present study, a coculture model of BM mesenchymal stromal cells (MSCs) and AML cells (KG1a cell-line and primary BM blasts) was used to explore this metabolic pathway. MSC-contact, rather than culture with MSC-conditioned medium, decreases ROS levels and inhibits the Nrf-2 pathway through overexpression of GPx3 in AML cells. The decrease of ROS levels also inactivates p38MAPK and reduces the proliferation of AML cells. Conversely, contact with AML cells modifies MSCs in that they display an increased oxidative stress and Nrf-2 activation, together with a concomitant lowered expression of GPx-3. Altogether, these experiments suggest that a reciprocal control of oxidative metabolism is initiated by direct cell-cell contact between MSCs and AML cells. GPx-3 expression appears to play a crucial role in this cross-talk and could be involved in the regulation of leukemogenesis.

Characteristics, combinations, treatments, and survival of second primary hematological neoplasm: a retrospective single-center cohort of 49 patients (Hemo2study).

The coexistence of dual hematological neoplasms is very rare. Sequential or synchronous neoplasms in hematology are an uncommon and complex clinical situation. The aim of the Hemo2 study was to describe the clinical characteristics and analyze the outcome of these patients. We performed a retrospective review of all patients diagnosed with sequential or synchronous hematological malignancies in the university hospital of Tours, between 2007 and 2018. We identified 49 patients in our study, with a prevalence of 0.89%. Sequential and synchronous combinations were found in 36 (73%) and 13 (27%) patients, respectively. One patient presented three sequential neoplasms. The median cumulative incidence was 6 years (95% CI 3-7). Among all neoplasms diagnosed (n = 99), we found 79 lymphoid neoplasms (LNs) (80%) and 20 myeloid neoplasms (MNs) (20%). Sex ratio was 1.88 with 65% of males and 35% of females. The most common LNs were Hodgkin lymphoma (n = 16; 16%) and multiple myeloma (n = 11; 11%). The most frequent MN was essential thrombocythemia (n = 5; 5%). The most common combination was Hodgkin lymphoma and follicular lymphoma in five (10%) patients. The overall survival from the first diagnosis (OS1) at 5 years was 82.4% (95% CI 72.1-94.3). The median overall survival from the second diagnosis (OS2) was 98 months (95% CI 44-NR) and 5-year OS2 was 58.7% (95% CI 45.5-75.7). Median progression-free survival from the second diagnosis (PFS) was 47 months (95% CI 27-NR) with 5-year PFS of 49% (95% CI 35.9-67). OS and PFS did not statistically differ between synchronous and sequential dual neoplasms. In this cohort, that the death relative risk (RR) was significantly lower if the second neoplasm appeared after more than 4 years following the first diagnosis (OR 0.37 (95% CI 0.16-0.90)). The Hemo2study confirmed the rarity of dual hematological neoplasms. In this cohort, HL and FL were the most frequent combinations. Our results may support that synchronous and sequential dual neoplasms bear the same prognosis. Further studies are needed to better characterize these uncommon clinical situations.

n-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation.

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.

Merkel cell carcinomas infiltrated with CD33+ myeloid cells and CD8+ T cells are associated with improved outcome.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare tumor of the skin that has an aggressive behavior. Immunity is the main regulator of MCC development, and many interactions between lymphocytes and tumor cells have been proven. However, the impact of tumor-infiltrating myeloid cells needs better characterization. OBJECTIVE: To characterize tumor-infiltrating myeloid cells in MCC and their association with other immune effectors and patient outcome. METHODS: MCC cases were reviewed from an ongoing prospective cohort study. In all, 103 triplicate tumor samples were included in a tissue microarray. Macrophages, neutrophils, and myeloid-derived suppressor cells were characterized by the following markers: CD68, CD33, CD163, CD15, CD33, and human leukocyte antigen-DR. Associations of these cell populations with programmed cell death ligand 1 expression, CD8 infiltrates, and vascular density were assessed. Impact on survival was analyzed by log-rank tests and a Cox multivariate model. RESULTS: The median density of macrophages was 216 cells/mm2. CD68+ and CD33+ macrophage densities were associated with CD8+ T-cell infiltrates and programmed cell death ligand 1 expression. In addition, MCC harboring CD8+ T cell infiltrates and brisk CD33+ myeloid cell infiltrates were significantly and independently associated with improved outcomes (recurrence-free and overall survival). LIMITATIONS: Sampling bias and the retrospective design were potential study limitations. CONCLUSION: Infiltration of CD33+ myeloid cells and CD8+ T lymphocytes defines a subset of MCC associated with improved outcome.

Percutaneous grafting with bone marrow autologous concentrate for open tibia fractures: analysis of forty three cases and literature review.

PURPOSE: Tibial fractures are the most common lower limb fractures. Some criteria such as open fractures and increasing open stage are known to be associated with high delayed union and pseudarthrosis rate. In cases of delayed or nonunion, classical treatment is autologous cancelous bone graft which is associated with high morbidity rate. The ideal treatment would be a percutaneous harvesting and grafting technique. As bone marrow autologous concentrate (BMAC) presents both advantages, we evaluated this technique from 2002 to 2007. METHODS: This was a retrospective study of 43 cases of open tibial fractures with initial surgical treatment. The criteria of inclusion were open fracture and nonunion, delayed union or suspicion of delayed union. RESULTS: In 23 cases (53.5 %) BMAC was successful. The success group had received significantly more CFU-F than the failure group (469 vs 153.10(3), p = 0.013). A threshold of 360.10(3) CFU-F grafted could be established over which there was 100 % success. BMAC done before 110 days after fracture had 47 % success and BMAC done since 110 days after fracture had 73 % success. BMAC success rate decreased with increasing initial fracture skin open stage. There was no BMAC success in cases of a fracture with a remaining gap of more than 4 mm. We had no complications with the technique at the iliac harvesting zone and tibia injection point. CONCLUSION: BMAC is a technique that should be considered as one of the different alternatives for management of long-bone delayed and nonunion because of its effectiveness, low complication rate, preservation of bone stock and low cost.

Aberrant DNA methylation impacts HOX genes expression in bone marrow mesenchymal stromal cells of myelodysplastic syndromes and de novo acute myeloid leukemia.

DNA methylation, a major biological process regulating the transcription, contributes to the pathophysiology of hematologic malignancies, and hypomethylating agents are commonly used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). In these diseases, bone marrow mesenchymal stromal cells (MSCs) play a key supportive role through the production of various signals and interactions. The DNA methylation status of MSCs, likely to reflect their functionality, might be relevant to understand their contribution to the pathophysiology of these diseases. Consequently, the aim of our study was to analyze the modifications of DNA methylation profiles of MSCs induced by MDS or AML. MSCs from MDS/AML patients were characterized via 5-methylcytosine quantification, gene expression profiles of key regulators of DNA methylation, identification of differentially methylated regions (DMRs) by methylome array, and quantification of DMR-coupled genes expression. MDS and AML-MSCs displayed global hypomethylation and under-expression of DNMT1 and UHRF1. Methylome analysis revealed aberrant methylation profiles in all MDS and in a subgroup of AML-MSCs. This aberrant methylation was preferentially found in the sequence of homeobox genes, especially from the HOX family (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4, and HOXC6), and impacted on their expression. These results highlight modifications of DNA methylation in MDS/AML-MSCs, both at global and focal levels dysregulating the expression of HOX genes well known for their involvement in leukemogenesis. Such DNA methylation in MSCs could be the consequence of the malignant disease or could participate in its development through defective functionality or exosomal transfer of HOX transcription factors from MSCs to hematopoietic cells.

Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition.

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids.

Mesenchymal stem cells (MSC)-spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSC-spheroids began to shrink after 24 hours, the size of spheroids from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, like hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.

Correction: Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The original version of this Article omitted the following from the Acknowledgements: This research was also supported by grants to KZ (UL and L-CNRS). This has now been corrected in both the PDF and HTML versions of the Article.

Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells.

The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients.

BACKGROUND AIMS: Advances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources. METHODS: We obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit-fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels. RESULTS: MSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential. CONCLUSIONS: Trabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.

The role of platelet/endothelial cell adhesion molecule 1 (CD31) and CD38 antigens in marrow microenvironmental retention of acute myelogenous leukemia cells.

In acute myelogenous leukemia (AML), leukemic cell-microenvironment interactions within various niches (stromal/osteoblastic or sinusoidal endothelial cell niches) have a role in leukemia cell survival and drug resistance. The AML leukemic cells express platelet/endothelial cell adhesion molecule-1 (CD31) and CD38, two adhesion molecules that could interact with microenvironmental elements, i.e., CD31 on the surface of marrow endothelial cells (CD31/CD31 and CD38/CD31 interactions) and hyaluronate (CD38/hyaluronate interactions). We report a physical association of these two antigens on the plasma membrane of myeloid leukemic cells. In this context, in vitro experiments done using interaction-blocking anti-CD31 and anti-CD38 monoclonal antibodies (CLB-HEC75 and OKT10, respectively) indicate that an excess of CD31 on the cell membrane of leukemic cells (CD31/CD38 MFI ratio >1) promotes a homotypic interaction with marrow endothelial cells, resulting in higher transendothelial migration. Conversely, an excess of CD38 (CD31/CD38 MFI ratio <1) allows leukemic cells to be entrapped within the bone marrow microenvironment through hyaluronate adhesion. The results obtained in vitro using fluorescence resonance energy transfer, co-capping, and co-immunoprecipitation experiments, and hyaluronate adhesion and transendothelial migration assays, are supported by immunophenotypic characterization of marrow leukemic cells from 78 AML patients on which CD38 expression levels were found to be positively correlated with those of CD31. Importantly, the excess of CD31 in those samples was associated with a higher peripheral WBC count. These findings indicate that bone marrow retention of AML cells depends on CD31 and CD38 coexpression levels.

Bone marrow oxidative stress and specific antioxidant signatures in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders with an inherent tendency for transformation in secondary acute myeloid leukemia. This study focused on the redox metabolism of bone marrow (BM) cells from 97 patients compared with 25 healthy controls. The level of reactive oxygen species (ROS) was quantified by flow cytometry in BM cell subsets as well as the expression level of 28 transcripts encoding for major enzymes involved in the antioxidant cellular response. Our results highlight increased ROS levels in BM nonlymphoid cells and especially in primitive CD34posCD38low progenitor cells. Moreover, we identified a specific antioxidant signature, dubbed "antioxidogram," for the different MDS subgroups or secondary acute myeloblastic leukemia (sAML). Our results suggest that progression from MDS toward sAML could be characterized by 3 successive molecular steps: (1) overexpression of enzymes reducing proteic disulfide bonds (MDS with <5% BM blasts [GLRX family]); (2) increased expression of enzymes detoxifying H2O2 (MDS with 5% to 19% BM blasts [PRDX and GPX families]); and finally (3) decreased expression of these enzymes in sAML. The antioxidant score (AO-Score) defined by logistic regression from the expression levels of transcripts made it possible to stage disease progression and, interestingly, this AO-Score was independent of the revised International Scoring System. Altogether, this study demonstrates that MDS and sAML present an important disturbance of redox metabolism, especially in BM stem and progenitor cells and that the specific molecular antioxidant response parameters (antioxidogram, AO-Score) could be considered as useful biomarkers for disease diagnosis and follow-up.

Mobile Exergaming in Adolescents' Everyday Life-Contextual Design of Where, When, with Whom, and How: The SmartLife Case.

Exergames, more specifically console-based exergames, are generally enjoyed by adolescents and known to increase physical activity. Nevertheless, they have a reduced usage over time and demonstrate little effectiveness over the long term. In order to increase playing time, mobile exergames may increase potential playing time, but need to be engaging and integrated in everyday life. The goal of the present study was to examine the context of gameplay for mobile exergaming in adolescents’ everyday life to inform game design and the integration of gameplay into everyday life. Eight focus groups were conducted with 49 Flemish adolescents (11 to 17 years of age). The focus groups were audiotaped, transcribed, and analyzed by means of thematic analysis via Nvivo 11 software (QSR International Pty Ltd., Victoria, Australia). The adolescents indicated leisure time and travel time to and from school as suitable timeframes for playing a mobile exergame. Outdoor gameplay should be restricted to the personal living environment of adolescents. Besides outdoor locations, the game should also be adaptable to at-home activities. Activities could vary from running outside to fitness exercises inside. Furthermore, the social context of the game was important, e.g., playing in teams or meeting at (virtual) meeting points. Physical activity tracking via smart clothing was identified as a motivator for gameplay. By means of this study, game developers may be better equipped to develop mobile exergames that embed gameplay in adolescents’ everyday life.

CXCL12 polymorphism and malignant cell dissemination/tissue infiltration in acute myeloid leukemia.

Stromal cell-derived factor 1 (SDF-1), a chemokine abundantly produced by the bone marrow microenvironment, and its receptor CXCR4 have crucial roles in malignant cell trafficking. In acute myeloid leukemia (AML), blasts invade the bloodstream and may localize in extramedullar sites, with variations from one patient to another. We hypothesized that a polymorphism in the SDF-1 coding gene (CXCL12 G801A) could influence blast dissemination and tissue infiltration in AML. CXCL12 G801A polymorphism was determined in 86 adult patients and 100 healthy volunteers. The allelic status and CXCR4 expression on bone marrow blasts were analyzed in relation to peripheral blood blast (PBB) counts and frequency of extramedullar tumor sites. 801A carrier status (801G/A, 801A/A) was found to be associated with a higher PBB count compared with 801G/G homozygous patients (P=0.031) and higher frequency of extramedullar tumor sites (odds ratio 2.92, 95% confidence interval 1.18-7.21, P=0.018). Moreover, the PBB count was correlated with CXCR4 expression (correlation coefficient 0.546, P=0.001) when considering 801A carriers. In conclusion, a polymorphism in the SDF-1 gene is shown for the first time to be associated with the clinical presentation of a malignant hematological disease and more generally with the risk of distant tissue infiltration by tumor cells.

Oncogenic STAT5 signaling promotes oxidative stress in chronic myeloid leukemia cells by repressing antioxidant defenses.

STAT5 transcription factors are frequently activated in hematopoietic neoplasms and are targets of various tyrosine kinase oncogenes. Evidences for a crosstalk between STAT5 and reactive oxygen species (ROS) metabolism have recently emerged but mechanisms involved in STAT5-mediated regulation of ROS still remain elusive. We demonstrate that sustained activation of STAT5 induced by Bcr-Abl in chronic myeloid leukemia (CML) cells promotes ROS production by repressing expression of two antioxidant enzymes, catalase and glutaredoxin-1(Glrx1). Downregulation of catalase and Glrx1 expression was also observed in primary cells from CML patients. Catalase was shown not only to reduce ROS levels but also, to induce quiescence in Bcr-Abl-positive leukemia cells. Furthermore, reduction of STAT5 phosphorylation and upregulation of catalase and Glrx1 were also evidenced in leukemia cells co-cultured with bone marrow stromal cells to mimic a leukemic niche. This caused downregulation of ROS levels and enhancement of leukemic cell quiescence. These data support a role of persistent STAT5 signaling in the regulation of ROS production in myeloid leukemias and highlight the repression of antioxidant defenses as an important regulatory mechanism.

Alteration Analysis of Bone Marrow Mesenchymal Stromal Cells from De Novo Acute Myeloid Leukemia Patients at Diagnosis.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) frequently display alterations in several hematologic disorders, such as acute lymphoid leukemia, acute myeloid leukemia (AML), and myelodysplastic syndromes. In acute leukemias, it is not clear whether MSC alterations contribute to the development of the malignant clone or whether they are simply the effect of tumor expansion on the microenvironment. We extensively investigated the characteristics of MSCs isolated from the BM of patients with de novo AML at diagnosis (L-MSCs) in terms of phenotype (gene and protein expression, apoptosis and senescence levels, DNA double-strand break formation) and functions (proliferation and clonogenic potentials, normal and leukemic hematopoiesis-supporting activity). We found that L-MSCs show reduced proliferation capacity and increased apoptosis levels compared with MSCs from healthy controls. Longer population doubling time in L-MSCs was not related to the AML characteristics at diagnosis (French-American-British type, cytogenetics, or tumor burden), but was related to patient age and independently associated with poorer patient outcome, as was cytogenetic prognostic feature. Analyzing, among others, the expression of 93 genes, we found that proliferative deficiency of L-MSCs was associated with a perivascular feature at the expense of the osteo-chondroblastic lineage with lower expression of several niche factors, such as KITLG, THPO, and ANGPT1 genes, the cell adhesion molecule VCAM1, and the developmental/embryonic genes, BMI1 and DICER1. L-MSC proliferative capacity was correlated positively with CXCL12, THPO, and ANGPT1 expression and negatively with JAG1 expression. Anyway, these changes did not affect their in vitro capacity to support normal hematopoiesis and to modify leukemic cell behavior (protection from apoptosis and quiescence induction). Our findings indicate that BM-derived MSCs from patients with newly diagnosed AML display phenotypic and functional alterations such as proliferative deficiency that could be attributed to tumor progression, but does not seem to play a special role in the leukemic process.

Bone marrow mesenchymal stromal cell (MSC) gene profiling in chronic myeloid leukemia (CML) patients at diagnosis and in deep molecular response induced by tyrosine kinase inhibitors (TKIs).

Although it has been well-demonstrated that bone marrow mesenchymal stromal cells (MSCs) from CML patients do not belong to the Ph1-positive clone, there is growing evidence that they could play a role in the leukemogenesis process or the protection of leukemic stem cells from the effects of tyrosine kinase inhibitors (TKIs). The aim of the present study was to identify genes differentially expressed in MSCs isolated from CML patients at diagnosis (CML-MSCs) as compared to MSCs from healthy controls. Using a custom gene-profiling assay, we identified six genes over-expressed in CML-MSCs (BMP1, FOXO3, MET, MITF, NANOG, PDPN), with the two highest levels being documented for PDPN (PODOPLANIN) and NANOG. To determine whether this aberrant signature persisted in patients in deep molecular response induced by TKIs, we analyzed MSCs derived from such patients (MR-MSCs). This analysis showed that, despite the deep molecular responses, BMP1, MET, MITF, NANOG, and PDPN mRNA were upregulated in MR-MSCs. Moreover, BMP1, MITF, and NANOG mRNA expressions in MR-MSCs were found to be intermediate between control MSCs and CML-MSCs. These results suggest that CML-MSCs exhibit an abnormal gene expression pattern which might have been established during the leukemogenic process and persist in patients in deep molecular response.

Influence of hypoxia on the domiciliation of mesenchymal stem cells after infusion into rats: possibilities of targeting pulmonary artery remodeling via cells therapies?

BACKGROUND: Bone marrow (BM) cells are promising tools for vascular therapies. Here, we focused on the possibility of targeting the hypoxia-induced pulmonary artery hypertension remodeling with systemic delivery of BM-derived mesenchymal stem cells (MSCs) into non-irradiated rats. METHODS: Six-week-old Wistar rats were exposed to 3-week chronic hypoxia leading to pulmonary artery wall remodeling. Domiciliation of adhesive BM-derived CD45- CD73+ CD90+ MSCs was first studied after a single intravenous infusion of Indium-111-labeled MSCs followed by whole body scintigraphies and autoradiographies of different harvested organs. In a second set of experiments, enhanced-GFP labeling allowed to observe distribution at later times using sequential infusions during the 3-week hypoxia exposure. RESULTS: A 30% pulmonary retention was observed by scintigraphies and no differences were observed in the global repartition between hypoxic and control groups. Intrapulmonary radioactivity repartition was homogenous in both groups, as shown by autoradiographies. BM-derived GFP-labeled MSCs were observed with a global repartition in liver, in spleen, in lung parenchyma and rarely in the adventitial layer of remodeled vessels. Furthermore this global repartition was not modified by hypoxia. Interestingly, these cells displayed in vivo bone marrow homing, proving a preservation of their viability and function. Bone marrow homing of GFP-labeled MSCs was increased in the hypoxic group. CONCLUSION: Adhesive BM-derived CD45- CD73+ CD90+ MSCs are not integrated in the pulmonary arteries remodeled media after repeated intravenous infusions in contrast to previously described in systemic vascular remodeling or with endothelial progenitor cells infusions.

Frequency and differentiation capacity of circulating LTC-IC mobilized by G-CSF or GM-CSF following chemotherapy: a comparison with steady-state bone marrow and peripheral blood.

OBJECTIVE: The present study was designed to compare directly the frequency of circulating LTC-IC and E-LTC-IC mobilized in peripheral blood (PB) after chemotherapy supported by either G-CSF (PB-G) or GM-CSF (PB-GM) in comparison to steady-state bone marrow (BM) and PB (PB-ST) values in the same patients. MATERIALS AND METHODS: Long-term cultures (LTC) were performed from 20 patients with malignant lymphoma at saturating cell concentrations to assess bulk progenitor cell production and by limiting dilution assay (LDA) to measure both frequency of LTC-IC and their proliferative and differentiation capacities. RESULTS: While CFC production in bulk LTC was higher at weeks 3-5 with PB-G than with PB-GM samples, week-5 LTC-IC and week-10 LTC-IC (E-LTC-IC) frequencies were not different using a LDA. However, the number of CFC derived from a single LTC-IC was higher in PB-G patients than in PB-GM patients (p = 0.01). Interestingly, the frequency of LTC-IC per 1 x 10(5) MNC in mobilized PB positively correlated with one-year marrow progenitor cell recovery, in contrast to the number of autografted CD34(+) cells and CFU-GM per kg. CONCLUSION: Both G-CSF and GM-CSF resulted in similar increases in LTC-IC and E-LTC-IC in PB at comparable levels to those present in BM. However, the differentiation capacity of LTC-IC was higher after mobilization with G-CSF than with GM-CSF, suggesting qualitative differences in LTC-IC mobilized with these growth factors.