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Pierre Charbord

Publications and source records attributed to Pierre Charbord.

15 recordsLinked to original sources
PubMed2006

Molecular profile of mouse stromal mesenchymal stem cells.

We determined a transcriptional profile specific for clonal stromal mesenchymal stem cells from adult and fetal hematopoietic sites. To identify mesenchymal stem cell-like stromal cell lines, we evaluated the adipocytic, osteoblastic, chondrocytic, and vascular smooth muscle differentiation potential and also the hematopoietic supportive (stromal) capacity of six mouse stromal cell lines from adult bone marrow and day 14.5 fetal liver. We found that two lines were quadripotent and also supported hematopoiesis, BMC9 from bone marrow and AFT024 from fetal liver. We then ascertained the set of genes differentially expressed in the intersection set of AFT024 and BMC9 compared with those expressed in the union set of two negative control lines, 2018 and BFC012 (both from fetal liver); 346 genes were upregulated and 299 downregulated. Using Ingenuity software, we found two major gene networks with highly significant scores. One network contained downregulated genes that are known to be implicated in osteoblastic differentiation, proliferation, or transformation. The other network contained upregulated genes that belonged to two categories, cytoskeletal genes and genes implicated in the transcriptional machinery. The data extend the concept of stromal mesenchymal stem cells to clonal cell populations derived not only from bone marrow but also from fetal liver. The gene networks described should discriminate this cell type from other types of stem cells and help define the stem cell state.

Animals↗
PubMed2006

Multipotential mesenchymal stem cells are mobilized into peripheral blood by hypoxia.

MSCs constitute a population of multipotential cells giving rise to adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells. It remains unclear whether MSCs can be isolated from adult peripheral blood under stationary conditions and whether they can be mobilized in a way similar to hematopoietic stem cells. In this report, we show that MSCs are regularly observed in the circulating blood of rats and that the circulating MSC pool is consistently and dramatically increased (by almost 15-fold) when animals are exposed to chronic hypoxia. The immunophenotype and the adipocytic, osteoblastic, and chondrocytic differentiation potential of circulating MSCs were similar to those of bone marrow MSCs. Hypoxia-induced mobilization appears to be specific for MSCs since total circulating hematopoietic progenitor cells were not significantly increased. Our data provide an in vivo model amenable to analysis of MSC-mobilizing factors.

Adipogenesis↗
PubMed2005

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.

Animals↗
PubMed2005

Gene expression in stem cell-supporting stromal cell lines.

Cells in the immediate microenvironment together with hematopoietic stem cells (HSCs) constitute the stem cell niche. The microenvironmental or stromal cells provide a complex molecular milieu that helps mediate and balance the self-renewal and commitment potentials of stem cells. The molecules in this milieu are not well defined. In this study, we have intersected previous cDNA subtraction studies with array expression methodologies to define and categorize known gene products expressed by HSC-supportive stromal cell lines. Data were curated from our previously released Stromal Cell Database (StroCDB) containing a set of gene products enriched for expression in the fetal liver stromal cell line AFT024. Global expression analyses were extended to other stem cell-supporting and -nonsupporting fetal liver stromal cell lines using commercially available microarrays. Known and previously described gene products from selected categories were studied: transcription factors, cell membrane proteins, cytoskeleton and related proteins, extracellular matrix proteins, cell adhesion molecules and their corresponding signaling molecules, and cytokines and their related mediators. More than 300 known gene products were selected for expression in HSC-supporting stromal cells compared to nonsupporting lines. Analyses of the data suggest that HSC-supportive cells are immature, sessile, and highly reactive after binding to integrin ligands and cytokines. Therefore, they provide a dynamic space poised to respond to molecular cues elaborated within the stem cell niche. The study provides a survey of known proteins that play key roles in the support of HSCs by fetal liver stromal cells. It also provides insights into the biology of the stem cell niche by highlighting the complex network of intercellular signaling and communication involved in the organization of the niche space.

Animals↗
PubMed2004

In vivo MR imaging of intravascularly injected magnetically labeled mesenchymal stem cells in rat kidney and liver.

PURPOSE: To evaluate in vivo magnetic resonance (MR) imaging with a conventional 1.5-T system for depiction and tracking of intravascularly injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs). MATERIALS AND METHODS: This study was conducted in accordance with French law governing animal research and met guidelines for animal care and use. Rat MSCs were labeled with SPIO and transfection agent. Relaxation rates at 1.5 T, cell viability, proliferation, differentiation capacity, and labeling stability were assessed in vitro as a function of SPIO concentration. MSCs were injected into renal arteries of healthy rats (labeled cells in four, unlabeled cells in two) and portal veins of rats treated with carbon tetrachloride to induce centrolobular liver necrosis (labeled cells and unlabeled cells in two each). Follow-up serial T2*-weighted gradient-echo MR imaging and R2* mapping were performed. MR imaging findings were compared histologically. RESULTS: SPIO labeling caused a strong R2* effect that increased linearly with iron dose; R2* increase for cells labeled for 48 hours with 50 microg of iron per milliliter was 50 sec(-1) per million cells per milliliter. R2* was proportional to iron load of cells. SPIO labeling did not affect cell viability (P > .27). Labeled cells were able to differentiate into adipocytes and osteocytes. Proliferation was substantially limited for MSCs labeled with 100 microg Fe/mL or greater. Label half-life was longer than 11 days. In normal kidneys, labeled MSCs caused signal intensity loss in renal cortex. After labeled MSC injection, diseased liver had diffuse granular appearance. Cells were detected for up to 7 days in kidney and 12 days in liver. Signal intensity loss and fading over time were confirmed with serial R2* mapping. At histologic analysis, signal intensity loss correlated with iron-loaded cells, primarily in renal glomeruli and hepatic sinusoids; immunohistochemical analysis results confirmed these cells were MSCs. CONCLUSION: MR imaging can aid in monitoring of intravascularly administered SPIO-labeled MSCs in vivo in kidney and liver.

Adipocytes↗
PubMed2004

Homing of in vitro expanded Stro-1- or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment.

The Stro-1 antigen potentially defines a mesenchymal stem cell (MSC) progenitor subset. We here report on the role of human ex vivo-expanded selected Stro-1(+) or Stro-1(-) MSC subsets on the engraftment of human CD34(+) cord blood cells in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. The data show that cotransplantation of expanded Stro-1(-) cells with CD34(+) cells resulted in a significant increase of human CD45, CD34, CD19, and CD11b cells detected in blood or in bone marrow (BM) and spleen as compared with the infusion of CD34(+) cells alone. Infusion into mice of expanded Stro-1(+) and Stro-1(-) cells (without CD34(+) cells) showed that the numbers of Stro-1(+)-derived (as assessed by DNA analysis of human beta-globin with quantitative polymerase chain reaction [PCR]) were higher than Stro-1(-)-derived cells in spleen, muscles, BM, and kidneys, while more Stro-1(-)-derived than Stro-1(+)-derived cells were found in lungs. The transduction of expanded Stro-1(+) cells with an enhanced green fluorescent protein (eGFP) gene did not modify their cytokine release and their homing in NOD/SCID mouse tissues. The difference between the hematopoietic support and the homing capabilities of expanded Stro-1(+) and Stro-1(-) cells may be of importance for clinical therapeutic applications: Stro-1(+) cells may rather be used for gene delivery in tissues while Stro-1(-) cells may rather be used to support hematopoietic engraftment.

Animals↗
PubMed2003

Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells.

Endothelial progenitor cells (EPCs) were shown to be present in systemic circulation and cord blood. We investigated whether EPCs display specific properties compared with mature endothelial cells. Human cord blood CD34+ cells were isolated and adherent cells were amplified under endothelial conditions. Expression of specific markers identified them as endothelial cells, also called endothelial progenitor-derived cells (EPDCs). When compared to mature endothelial cells, human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (HBMECs), endothelial markers, were expressed to the same extent except for KDR, which is expressed more in EPDCs. They display a higher proliferation potential. Functional studies demonstrated that EPDCs were more sensitive to angiogenic factors, which afford these cells greater protection against cell death compared with HUVECs. Moreover, EPDCs exhibit more hematopoietic supportive activity than HUVECs. Finally, studies in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice demonstrated that human circulating EPCs are able to colonize a Matrigel plug. EPDCs display the morphology and phenotype of endothelial cells. Their functional features indicate, however, that although these cells have undergone some differentiation steps, they still have the properties of immature cells, suggesting greater tissue repair capabilities. Future use of in vitro amplified peripheral blood EPDCs may constitute a challenging strategy for cell therapy.

Animals↗
PubMed2003

Stromal-derived factor 1 and matrix metalloproteinase 9 levels in bone marrow and peripheral blood of patients mobilized by granulocyte colony-stimulating factor and chemotherapy. Relationship with mobilizing capacity of haematopoietic progenitor cells.

The roles of the chemokine stromal-derived factor 1 (SDF-1) and the matrix metalloproteinase 9 (MMP-9) in haematopoietic progenitor cell (HPC) mobilization are still unclear, particularly when patients are mobilized by granulocyte colony-stimulating factor (G-CSF) plus chemotherapy. We determined bone marrow (BM) and peripheral blood (PB) plasma levels of SDF-1, together with CXC-chemokine receptor 4 (CXCR-4) expression on CD34+ cells, and interleukin 8 (IL-8) and MMP-9 in 55 patients mobilized for autologous PB transplantation compared with 10 normal BM and PB samples. Plasma samples were tested at steady state (SS-) and after mobilization by cyclophosphamide and G-CSF administration (M-). SDF-1, CXCR-4, IL-8 and MMP-9 levels were significantly lower in SS- and M-PB than in SS-BM. Differences in SDF-1 levels between SS-PB and SS-BM were also observed after mobilization. We showed for the first time a clear relationship between the levels of circulating HPC, both at steady state and after mobilization, and those of secreted MMP-9 but not of SDF-1 or IL-8. However, a negative correlation was observed between mobilizing capacity and CXCR-4 expression on CD34+ cells. These findings suggest that G-CSF-induced mobilization of HPC from BM involves MMP-9, without reversing the positive gradient of SDF-1 between BM and PB.

Adult↗
PubMed2002

Fetal liver stroma consists of cells in epithelial-to-mesenchymal transition.

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work, we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin, osteopontin, collagen I, alpha smooth muscle actin, thrombospondin-1, EDa fibronectin, calponin, Stro-1 antigens, myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein, cytokeratins 8 and 18, albumin, E-cadherin, hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT), often observed during development, including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation, induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment.

Animals↗
PubMed2002

A molecular profile of a hematopoietic stem cell niche.

The hematopoietic microenvironment provides a complex molecular milieu that regulates the self-renewal and differentiation activities of stem cells. We have characterized a stem cell supportive stromal cell line, AFT024, that was derived from murine fetal liver. Highly purified in vivo transplantable mouse stem cells are maintained in AFT024 cultures at input levels, whereas other primitive progenitors are expanded. In addition, human stem cells are very effectively supported by AFT024. We suggest that the AFT024 cell line represents a component of an in vivo stem cell niche. To determine the molecular signals elaborated in this niche, we undertook a functional genomics approach that combines extensive sequence mining of a subtracted cDNA library, high-density array hybridization and in-depth bioinformatic analyses. The data have been assembled into a biological process oriented database, and represent a molecular profile of a candidate stem cell niche.

Amino Acid Sequence↗
PubMed2002

Comparative study of stromal cell lines derived from embryonic, fetal, and postnatal mouse blood-forming tissues.

OBJECTIVE: To better understand the differentiation of stromal cells of the hematopoietic microenvironment, we set out to characterize stromal cells from the different developmental sites of hematopoiesis in the mouse (30 bone marrow, 7 spleen, 3 embryonic and 15 fetal liver, 6 yolk sac, and 6 aorta-gonad-mesonephros lines) for expression of 22 cytoskeletal, membrane, and extracellular matrix proteins. MATERIALS AND METHODS: Western blotting, immunofluorescence, and flow cytometry were used. Statistical methods included principal components analysis and analysis of variance. RESULTS: Stromal cells from 11 dpc mouse embryos express mesenchymal and vascular smooth muscle cell (VSMC) markers. Principal components analysis on the 70 stromal cell lines isolated from different anatomic sites and developmental stages allows classification of stromal lines along a mesenchymal to VSMC differentiation pathway. Stromal cells do not express endothelial and hematopoietic differentiation membrane antigens, but they do express integrin alpha(5), alpha(6), and beta(1) subunits, vascular cell adhesion molecule-1, CD44, stem cell antigen-1, Thy-1, CD34, and endoglin. The intensity of expression of certain markers differs between lines according to the anatomic site of origin. CONCLUSIONS: This study indicates that stromal cells, whatever their anatomic site of origin, follow a VSMC differentiation pathway, suggesting a blood-forming tissue-specific differentiation of mesenchymal stem cells. Differential quantitative expression of distinct sets of markers appears to be correlated with the anatomic sites of origin of the stromal cells.

Aging↗
PubMed2002

Human bone marrow angiogenesis: in vitro modulation by substance P and neurokinin A.

We have previously described a culture system for human bone marrow endothelial cells that organize into capillary tubes associated to pericytes. In the present work, we used this model to assess the angiogenic properties of tachykinins, which have been demonstrated to be involved in neuro-immuno-haematopoietic interactions. The substance P (SP) and neurokinin A (NKA) were similarly potent at increasing in vitro angiogenesis, via NK1 and NK2 receptors respectively. These mediators were not produced by cells in culture, suggesting that in vivo they may be released by nerve fibres in the bone marrow. Therefore, we looked for in situ innervation of the human bone marrow, unknown to date, using immunohistochemistry techniques. As in rodents, arterioles were largely innervated, associated with between one and 10 nerve fibres. Capillary innervation was more restrictive as a unique thin nerve fibre was found in the vicinity of only 6% of these vessels. Finally, no nerve fibres were observed in the vicinity of sinus walls. In conclusion, both in vitro results and the anatomical display of nerve fibres suggest a role in human bone marrow for the vasoactive neuropeptides SP and NKA, which were secreted into a perivascular location. These neural mediators might modulate blood flow in the bone marrow both in the short term by adjusting vascular tone and in the long term by inducing angiogenesis.

Angiogenesis Inducing Agents↗
PubMed2002

The STRO-1+ marrow cell population is multipotential.

Human marrow-derived STRO-1+ cells were tested for their ability to differentiate into multiple mesenchymal phenotypes. STRO-1+ cells were isolated from long-term cultures of human marrow cells by magnetic immunobeads linked to the antibody STRO-1. Stromal layers generated from STRO-1+ cells, when cocultured without exogenous cytokines for several weeks with CD34+/CD38+ and CD34+/CD38low cells, were able to induce a 10- to 20-fold increase in colony-forming units, and to sustain cobblestone area colony-forming cells with a distinct time course for the different hematopoietic precursors. A myofibroblastic and adipocytic phenotype was observed in hematopoiesis-supportive stromal cells, as shown by electron microscopy and staining of lipids with Nile red. The adipocytic potential of STRO-1+ cells was further demonstrated by flow cytometry of Nile red O-stained cells cultured in adipocyte-induced medium. The same population of STRO-1+ cells cultured in osteoblast-inducing medium was shown to have osteogenic potential, as demonstrated in vitro by the formation of mineral (von Kossa staining), and in vivo by the formation of bone tissue within porous calcium phosphate ceramics implanted into athymic mice. This population of STRO-1+ cells was also shown to have chondrogenic potential in aggregate cultures which displayed a morphologic appearance of cartilage and stained positively with antibodies specific to type II and type X collagens. These results show that the subset of marrow cells that express the STRO-1 antigen is capable of differentiating into multiple mesenchymal lineages including hematopoiesis-supportive stromal cells with a vascular smooth muscle-like phenotype, adipocytes, osteoblasts and chondrocytes.

ADP-ribosyl Cyclase↗
PubMed2002

Origin and differentiation of human and murine stroma.

Stromal cells generated in long-term cultures appear to follow a vascular smooth muscle differentiation pathway. Such a pathway, comprising several steps hallmarked by the expression of cytoskeletal and extracellular matrix markers, is found not only for bone marrow stromal cells, but also for stromal cells generated from the different developmental sites of hematopoiesis (yolk sac, aorta-gonad-mesonephros region, fetal liver, and spleen). Factors responsible for this differentiation pathway and its functional significance are discussed. The mesenchymal founder cell might be, at least for bone marrow, a mesenchymal stem cell (MSC), giving rise to stromal cells, endothelial cells, adipocytes, osteoblasts, and chondrocytes. A feature that distinguishes the MSC lineage from that of the hematopoietic stem cell lineage is that differentiation pathways are not strictly delineated, since even apparently fully differentiated cells from a given lineage have the potential to convert into another lineage (phenotype "plasticity") and intermediate cell phenotypes are observed. A stochastic Repression/Induction model that would account for this plasticity is proposed.

Animals↗