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We have studied the plasma membrane protein phenotype of human culture-amplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in colony-forming units–fibroblasts was low for CD49b, CD90, and CD105, but high for CD73, CD130, CD146, CD200, and integrin alphaV/beta5. In addition, the expression of CD73, CD146, and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in-depth studies.

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 β-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. (Blood. 2004;103:3313-3319)

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 β-globinwith 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. (Blood. 2004;103:3313-3319)

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.

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, α smooth muscle actin, thrombospondin-1, EDa fibronectin, calponin, Stro-1 antigens, myocyte-enhancer factor 2C) and epithelial (α-fetoprotein, cytokeratins 8 and 18, albumin, E-cadherin, hepatocyte nuclear factor 3 α) 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.

CD40 ligand (CD40L)/CD40 interactions play a central role in T-cell–dependent B-cell activation as previously shown by in vitro studies, the phenotype of CD40L knockout mice and the defective expression of CD40L in patients who have X-linked immunodeficiency with hyper-IgM. The distribution of CD40 in cells other than of myeloid and lymphoid lineages has suggested additional functions for this receptor/ligand couple. Here we show that CD40L stimulates myelopoiesis with a noticeable effect on megakaryocytopoiesis in cocultures of hematopoietic progenitor cells and bone marrow stromal cells. These results suggest a mechanism by which T-cell or platelet-associated or soluble CD40L may regulate myelopoiesis.

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38−/lo, Thy-1+, rho123lo, CD34+hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1+progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34+CD38−hematopoietic precursors, and CD3+CD6+peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34+rho123med/loprogenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38-/lo, Thy-1+, rho123lo, CD34+hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1+ progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34+ CD38-hematopoietic precursors, and CD3+ CD6+peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34+ rho123med/lo progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.

The CGA-7, a monoclonal antibody that reacts with smooth muscle cell actin but not with endothelial cell or fibroblast actin, and HHF, a monoclonal antibody that reacts with smooth muscle, skeletal muscle, and cardiac muscle actin, both recognize microfilaments present within adherent cells from actively hematopoietic human long-term marrow cultures. Macrophages, monocytes, and cultured marrow fibroblasts do not react with either antibody. These data suggest that the anti-actin antibodies may serve as useful markers for in vitro microenvironmental cells and lend support to the hypothesis that stromal cells from long- term marrow cultures are different from marrow fibroblasts and may constitute a unique cell lineage.

Adherent cells from long-term marrow cultures from 23 individuals were transformed with wild-type simian virus 40 (SV40). After transformation, cloned cell lines were developed that even after rigorous subcloning invariably produced both stromal cells and round cells. The stromal cells expressed cytoskeletal filaments similar to those of long-term marrow culture adherent cells and produced interstitial and basal lamina collagen types. The round cells had the electron microscopic appearance of primitive hematopoietic cells and when examined with cytochemical stains and monoclonal antibodies to hematopoietic differentiation antigens had reaction patterns suggestive of cells from several lineages. Most round cells expressed the pan- hematopoietic T-200 determinant, and lesser percentages expressed the early T cell antigens CD-1 and CD-3, HLA-DR determinants, the monocytic antigen recognized by Leu M3, and the myeloid antigens detected by monoclonal antibodies 1G10 and 12.8. In addition, when plated in semisolid medium in the presence of a source of colony-stimulating activity, up to 11% of the cells formed colonies consisting of blastlike cells that also expressed hematopoietic cell surface determinants. The data suggest that adherent cells in long-term marrow cultures contain a cell that after transformation by SV40 obligately produces cells with hematopoietic as well as stromalike features.

No relevant conflicts of interest to declare.

Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitro migration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2 for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitro and for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.

Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitromigration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitroand for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.

The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitro migration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+ cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+ cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitro trans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+ across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.

The mechanism of G-CSF-induced hematopoietic progenitor/stem cell (HPC/HSC) mobilization from bone marrow microenvironment to peripheral blood has been related to local production by neutrophils of proteases, including elastase, cathepsin G and metalloproteinases (MMPs). We previously showed that in vitromigration of hematopoietic cells across a layer of human marrow stromal cells (MSC) is induced when stimulated by G-CSF. In the present study, we investigated the role of MMPs produced by MSC used for the trans-stromal migration of MO7e line and marrow CD34+cells. Normal human MSC were previously grown to confluence on Transwell® filters (pore diameter 5-μm) and then stimulated or not by IL-1 (15 U/mL/Day) or G-CSF (15 ng/mL/Day) for three consecutive days. In a second step, MO7e or marrow CD34+cells, put in the upper chamber, were allowed to migrate through the layers (4 hrs, SDF-1 100 ng/mL in the bottom chamber). The contribution of MMPs was evaluated in this in vitrotrans-stromal migration assay using specific blocking monoclonal antibodies (mAb), anti-MMP-1, anti-MMP-2 and anti-MMP-9 (each at 5 μg/mL). The amounts of antigenic MMP-2 and MMP-9 produced by MSC were evaluated in cell supernatants by enzyme-linked immunosorbent assay (ELISA) and gelatinolytic activity of MMPs by zymography. mRNA expression of MMPs in MSC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). None of the MSC layers used contained detectable hematopoietic cells since they were all CD45-. Migration of MO7e cells across unstimulated or G-CSF-stimulated stromal cells was significantly inhibited by specific mAb. Percentages of inhibition were 25+/−5 % and 54+/−9 % for anti-MMP-2 mAb, and 18+/−6 % and 53+/−9 % for anti-MMP-9 mAb, respectively. Using the combination of anti MMP-2 and anti-MMP-9, inhibition reached 71+/−24 % across stimulated layers. In contrast, anti-MMP-1 mAb did not show any significant inhibition. Migration of CD34+across G-CSF-stimulated stromal cells was similarly inhibited with percentages of 38+/−18 % for anti-MMP- 2 mAb, and 34+/−11 % for anti-MMP-9 mAb, while migration was not inhibited again, using anti-MMP-1 mAb. In parallel, we demonstrated that MSC did express G-CSF receptor by RT-PCR. Production of MMPs by unstimulated and G-CSF-stimulated MSC was detected by ELISA, zymography and RT-PCR for MMP-2, and by zymography and RT-PCR for MMP-9. In conclusion, G-CSF-induction of HPC trans-stromal migration involves MMP-2 and MMP-9 but not MMP-1. These findings suggest that microenvironment, in the absence of neutrophils, could have a significant role in G-CSF mobilization of HPC/HSC. A local production of MMP-2 and MMP-9 by marrow stromal cells could be critical in the egress of HSC out of the hematopoietic niche.