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THE STEM CELL NICHE

Human Bone Marrow Adipocytes Block Granulopoiesis Through Neuropilin-1-Induced Granulocyte Colony-Stimulating Factor Inhibition

Departments of ^aCytology and Histology and
^bPathological Anatomy (Groupe Interdisciplinaire de Génoprotéomique Appliquée-Recherche), University of Liege, Belgium;
^cCentre National de la Recherche Scientifique-Unité Mixte de Recherche, Université Paris V. René Descartes,
^gLaboratory of Hematology, Université Paris V. René Descartes, Assistance Publique-Hôpitaux de Paris, and
ⁱDepartment of Clinical Hematology, Université Paris V. René Descartes, Assistance Publique-Hôpitaux de Paris, Hôpital Necker, Paris, France;
^dService de Biochimie et Hormonologie, Hôpital Tenon, Paris, France;
Laboratories of ^eHematobiology and Immunohematology (Groupe Interdisciplinaire de Génoprotéomique Appliquée-Recherche) and
^fConnective Tissue Biology (Groupe Interdisciplinaire de Génoprotéomique Appliquée-Recherche), University of Liege, Liège, Belgium

Key Words. Adult human bone marrow • Granulopoiesis • Stem cell-microenvironment interactions • Bone marrow stromal cells • In vitro differentiation • Bone marrow adipocytes • Neuropilin-1 • Macrophages

Correspondence: Correspondence: Marie Paule Defresne, M.D., Ph.D., Department of Cytology and Histology (Groupe Interdisciplinaire de Génoprotéomique Appliquée-Recherche), University of Liege, CHU-B23, 4000 Liège, Belgium. Telephone: 32-4-366-29-19; Fax: 32-4-366-29-19; e-mail: mp.defresne{at}ulg.ac.be; or Olivier Hermine, M.D., Ph.D., Centre National de la Recherche Scientifique-Unité Mixte de Recherche 8147, Université Paris V. René Descartes, Hôpital Necker, 161 rue de Sèvres, 75015 Paris, France. Telephone: 0033144490675; Fax: 0033144490676; e-mail: hermine{at}necker.fr

Received on January 22, 2008; accepted for publication on March 24, 2008.

First published online in STEM CELLS EXPRESS  April 3, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34⁺ differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34⁺ cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 β or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The bone marrow (BM) microenvironment plays a critical role in regulating proliferation and differentiation of hematopoietic cells (HC) [1, 2]. This process depends on complex interactions between both compartments that involve growth factors and cytokines [3, 4], as well as direct cell-to-cell contacts through surface molecules [5–8]. In femoral, so-called fatty BM, the microenvironment of residual HCs, which include quiescent stem cells and macrophages (Mo), is composed mainly of adipocytes. Several nonhematopoietic functions of adipocytes have been reported, such as a contribution to systemic lipid metabolism, supply of organ-specific energy, and promotion of osteogenesis [9, 10]. Furthermore, adipocytes are regarded as part of an endocrine organ [11, 12], since they produce hormones such as leptin [13]. In contrast, up to now, in humans, their role in hematopoiesis has been questioned. The participation of adipocytes in the regulation of hematopoiesis is plausible because of their capacity to produce a number of molecules required in this process, including vascular endothelial growth factor A (VEGF-A) [14, 15], cytokines such as stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), and interleukin (IL)-6 [16, 17], and the nonclassic VEGF receptor known as neuropilin-1 (NP-1) [18].

NP-1 can bind two types of ligands, semaphorin of class-3 [19] and VEGF [20], both of which take part in regulating complex cell-to-cell interactions, including neuronal guidance [21], angiogenesis [22], and regulation of central and peripheral immunity [23, 24]. Neuropilin-1 has also been described as a functional cell-surface receptor in the murine MS5 stromal cell line and is involved in the overexpression of thrombospondin and fms-like tyrosine kinase-3 ligand, both of which regulate early events in hematopoiesis [25]. However, nothing is known about the involvement of NP-1 in late stages of differentiation.

We reported in a previous study that NP-1 expression was higher in femoral than in iliac crest BM and that it was associated mainly with adipocytes [18], raising the question of its physiologic relevance in hematopoiesis. To address this issue, we collected adipocytes from human femoral BM that acquired fibroblast-like fat cell (FLFC) morphology in vitro but conserved the typical features of freshly isolated bone marrow adipocytes, including expression of specific surface molecules such as leptin and preadipocyte factor-1 (Pref-1)/-like protein-1 (dlk-1).

Using FLFCs as a model of primary adipocytes, we examined the effects of FLFCs (or adipocytes) on CD34⁺ progenitor cell differentiation and the putative involvement of NP-1 in this process. We provide evidence that adipocytes participate in hematopoiesis by hampering granulopoiesis through inhibition of the production of granulocyte colony-stimulating factor (G-CSF), a cytokine required in polynuclear neutrophil (PNN) differentiation [26–28]. We show that this negative regulation occurs through cell-to-cell contact in which NP-1 plays a major role. We discuss the possible implication of these findings in pathological conditions.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Isolation and Culture of Adipocytes and Bone Marrow-Derived Stromal Cells
Femoral BM and iliac crest BM samples were obtained from patients undergoing hip surgery at the Department of Orthopedic Surgery of the University Hospital of Liege, Belgium. The ethical committee approved this study, and the donors were informed in accordance with the Helsinki convention. Cells from femoral biopsies were isolated by digestion with type II collagenase solution (3 mg/ml) (Sigma-Aldrich, St. Quentin le Fallavier, France, http://www.sigmaaldrich.com) for 1 hour 30 minutes at 37°C. Floating adipocyte were separated by centrifugation for 10 minutes at 700 rpm and then filtered on a 200-µm membrane. Adipocytes were cultured using the ceiling method as previously described [29, 30] and amplified using a long-term culture medium (-minimal essential medium, 12.5% horse serum, 12.5% fetal calf serum, 2 mM L-glutamine, 0.2 M inositol, and 100 M β-mercaptoethanol). Marrow-derived stromal cells (MDSCs) were obtained from iliac crest BM after Ficoll gradient (GE Healthcare, Uppsala, Sweden, http://www.gehealthcare.com) and seeding in a flask.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was purified from cell suspensions using the High Pure RNA Isolation kit (Roche Diagnostics GmbH, Mannheim, Germany, http://www.roche-applied-science.com). The amount of purified RNA was quantified by fluorimetry using the RiboGreen RNA Quantification kit (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). The following specific primers were used to amplify the following mRNA: endogens 28S, 3'-GTTCACCCACTAATAGGGAACGTGA and 5'-GATTCTGACTTAGAGGCGTTCAGT; glyceraldehyde-3-phosphate de-hydrogenase (GAPDH), 5'-CCTGGCCAAGGTCATCCATGACA and 3'-GGGATGACCTTGCCCACAGCCTT; glycerol-3-phosphate dehydrogenase (G3PDH), 5'-GTTATTGGAGGAGGAGCAACAGGA and 3'-CTAGCAGGTTGGCACGCTCATGA; adipocyte fatty acid-binding protein (aP2), 5'-GGTACCTGGAAACTTGTCTCCAGT and 3'-TCCTGTCATCTGCAGTGACTTCGT; lipoprotein lipase (LPL), 5'-GAC-TCGTTCTCAGATGCCCTACAA and 3'-CCACCAGTCTGACCAGCTAAAGTA; peroxisome proliferator-activated receptor 2 (PPAR2), 5'-CAGTGGGGATGTCTCATAATGCCA and 3'-TGTCTTTCCTGTCAAGATCGCCCT; G-CSF, 5'-AGCTTCCTGCTCAAGTGCTTAGAG and 3'-TTCTTCCAT-CTGCTGCCAGATGGT; granulocyte-macrophage colony-stimulating factor (GM-CSF), 5'-GTCTCCTGAACCTGAGTAGAGACA and 3'-AAGGGGATGACAAGCAGAAAGTCC.

Reactions were performed in an automated thermal cycler (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) using the geneAmp Thermostable rTth Reverse Transcriptase RNA polymerase chain reaction (PCR) kit (PerkinElmer), specific pairs of primers (Eurogentec, Seraing, Belgium, http://www.eurogentec.be), and 10 ng of RNA per 25 µl of reaction mixture. Known copy number of the internal standard (synthetic standard RNA) was used for GAPDH and 28S mRNA amplification.

Cocultures of CD34⁺ Progenitors with FLFCs or MDSCs
FLFCs and MDSCs were cultured in 6- or 24-well plates or in transwell chambers at 150 x 10³ cells or 30 x 10³ cells, respectively. After cell layer formation, 5 x 10³ (6-well plates) or 2 x 10³ (24-well plates) CD34⁺ cells enriched by magnetic bead separation kit according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) were added. Cocultures were stopped after 1–5 weeks for analysis. Conditioned media were collected and conserved at –80°C until analysis of leptin, G-CSF, and IL-1 by enzyme-linked immunosorbent assay (ELISA) (R&D Systems Inc., Lille, France, http://www.rndsystems.com). Cytospins were realized for May-Grünwald-Giemsa (MGG) staining. Flow cytometry analysis was performed on a FACSCalibur (Becton, Dickinson and Company, Lille, France, http://www.bd.com) after cell staining with a goat polyclonal-anti-NP-1 antibody followed by a fluorescein isothiocyanate-rabbit anti-goat IgG antibody.

FLFC and CD34⁺ Transplantation in NOD/SCID Mice
FLFCs (1 x 10⁶ cells) alone or added to 5 x 10³ CD34⁺ cells were grafted into the kidney capsule of 8-week-old NOD/SCID mice as previously described [31]. Fifteen days after transplantation, kidneys were collected, embedded in Tissue-Tek (Sakura Finetek, Europe B.V., Zoeterwoude, The Netherlands, http://www.sakura.com), and frozen at –80°C. Five-micrometer sections were cut and stained with hematoxylin and eosin or stained with anti-adiponectin (R&D Systems) and anti-Mac-387 (Dako N.V., Heverlee, France, http://www.dako.com). Staining was detected by horseradish peroxidase (HRP)-conjugated secondary antibody and the liquid Diaminobenzidine (DAB) system (Dako). Granulopoiesis was analyzed by chloroacetate esterase activity detection. Human BM specimen was used as a positive control.

Immunohistochemical Studies of Pref-1/d1k-1 and G-CSF Expression
The expression of Pref-1/dlk-1 was analyzed in paraffin-embedded human bone marrow, in human bone marrow-isolated adipocytes, and in cultured cells (FLFCs, mesenchymal stem cells [MSC], and HS5) using a goat anti-human antibody, DLK (N18) (1:5). The specificity of the staining was assessed by incubating the antibody with blocking peptide DLK (N-18)-P (1/1) (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). For tissue sections, dlk-1/Pref-1 was detected by using horseradish peroxidase-conjugated rabbit anti-goat IgG as secondary antibody revealed with liquid DAB system. For bone marrow-isolated adipocytes and cultured cells, we used a biotinylated rabbit anti-goat IgG as a secondary antibody (1:200) revealed with streptavidin-Alexa488 (1:500) (Invitrogen, Liège, Belgium, http://www.invitrogen.com). Tissue sections were deparaffined by xylene and hydrated by an ethanol gradient (100% to 20% vol/vol). The hydrated sections were incubated in 3% H₂O₂ to quench endogenous peroxidase activity. Slides were incubated with an unmasking EDTA solution (1 mM) (PROSAN; Dako), and nonspecific sites were blocked with 3% horse serum, 1% bovine serum albumin before being incubated with primary and secondary antibodies. Bone marrow adipocytes and cultured cells were first fixed with cold acetone for 5 minutes and treated for immunostaining as described above. For the G-CSF expression analysis by immunostaining in FLFCs/CD34⁺ and MDSCs/CD34⁺ in cell-cell contact and in transwell chambers cocultures, adherent macrophages alone or with FLFCs were fixed with cold acetone for 5 minutes, and endogenous peroxidases were blocked with H₂O₂ for 30 minutes. Cells were then stained by a goat anti-G-CSF (R&D Systems) in saponin buffer (1/50) for 1 hour at room temperature. A positive staining was detected by biotinylated rabbit anti-goat IgG as a secondary antibody (1:200) and revealed by streptavidin-HRP (1/100) and liquid DAB system (all from Dako N.V.).

Cocultures of Granulocyte Precursors with FLFCs and/or Monocytes
CD34⁺ were seeded at 5 x 10⁵ cells in 1 ml with SCF (50 ng/ml), IL-3 (10 ng/ml), GM-CSF (5 ng/ml), and G-CSF (10 ng/ml). At day 5, granulocyte precursors (PreGra) were enriched using the CD36⁺ and CD14⁺ monocyte depletion kit (Miltenyi Biotec). Polynuclear neutrophil maturation was analyzed after culture of 10⁴ PreGra with either 10⁴ monocytes or 25 x 10³ FLFCs. The cocultures were done in cell-cell contact or using 0.4-µm transwell chambers. Cocultures were stopped after 16 days and analyzed by MGG staining. The absolute number of mature granulocytes was evaluated as a mean of mature segmented PNN in a field of 100 cells of immature PreGra and mature segmented PNN excluding monocytes/macrophages and FLFCs.

Neutralization of Neuropilin-1
We used a neutralizing antibody (endotoxin-free polyclonal goat anti-NP-1 antibody) (a generous gift from A. Kolodkin, Johns Hopkins University) well reported by our group [23] and others [32, 33]. Zero to 20 µg/ml was added in FLFC and CD34⁺ cocultures for 5 weeks. This anti-NP-1 antibody was also added for 2 weeks in PreGra, macrophage, and FLFC coculture at 20 µg/ml.

Statistical Analysis
The results were analyzed by independent samples in two-tailed and unpaired Student's t tests and are presented as mean ± SE deviation.

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Fibroblast-Like Fat Cells as an Experimental Model to Investigate Adipocyte-Myelo-Monocytic Interactions in Femoral Bone Marrow
It was previously shown that relative to iliac crest BM, the femoral counterpart is enriched in adipocytes, and the incidence of hematopoietic cells is accordingly decreased. This observation raised the question of whether adipocytes exerted a negative control on differentiation from CD34⁺ progenitors. To address this issue, we used adipocytes isolated from femoral BM using the ceiling method [29, 30]. The cells obtained in these conditions exhibited cytological features of fibroblasts (Fig. 1Aa–1Ac). They had lost their big oil droplet but still contained small lipid droplets stained by Oil Red O (Fig. 1Ac) and could return to the unilocular stage in adipogenic medium (data not shown). To further investigate the similarity between FLFCs and freshly isolated adipocytes, we compared the expression profile of typical lineage-specific genes in both cell types, in the absence of exogenous factors, either alone or together with CD34⁺ cells, which are normally present in the microenvironment. The phenotype of FLFCs closely resembled that of mature BM fat cells. They expressed CD36 but lacked common hematopoietic markers, such as CD34, CD14, CD45, CD68, CD3, and CD8 (data not shown). Furthermore, transcripts encoding typical adipocyte markers, such as early LPL, intermediate PP(AR2), late aP2, and G3PDH, were easily detected by reverse transcription (RT)-PCR (Fig. 1B). Leptin, a very late marker of adipocyte differentiation, was present in culture supernatants, as assessed by ELISA (Fig. 1C). LPL and aP2 expression, as well as leptin production, were increased in FLFC/CD34⁺ cocultures relative to FLFCs alone (Fig. 1B, 1C). To further investigate the relationship between FLFCs and primary adipocytes, we analyzed the expression of Pref-1/dlk, a membrane protein regarded as a preadipocyte marker, because it has been shown to be downregulated during in vitro terminal differentiation [34].

View larger version (37K): [in this window] [in a new window]   Figure 1. Characterization of FLFCs. (A): Adipocytes in culture acquired an FLFC morphology (Aa–Ac) but still contained Oil Red O-positive droplets (Ac) and were stained with anti-NP-1 antibody (Ad). (B): Reverse transcription (RT)-polymerase chain reaction (PCR) was used to analyze the presence of adipogenic markers (LPL, aP2, G3PDH, PPAR2) in freshly isolated adipocytes (lane 1), FLFC/CD34+ cocultures (lane 2), and FLFCs (lane 3). Internal ssRNA and 28S oligos were used to verify RT-PCR efficiency and to standardize PCR product. (C): Leptin production assessed by enzyme-linked immunosorbent assay in FLFC– CD34+ cocultures and in cocultures of marrow stroma and CD34+. (D): Immunohistochemistry detected preadipocyte factor-1 (Pref-1)/-like protein-1 (dlk-1)-positive adipocytes in the bone marrow (Da), and the specificity was tested using blocking peptide (Db). (E): Freshly isolated marrow adipocytes (Ea) and FLFCs (Eb) expressed Pref-1/dlk-1; HS5 (human stromal cell line) (Ec), and mesenchymal stem cells (Ed) were negative. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. Data are representative of one of three independent experiments. Abbreviations: aP2, adipocyte fatty acid-binding protein; FLFC, fibroblast-like fat cells; G3PDH, glycerol-3-phosphate dehydrogenase; LPL, lipoprotein lipase; NP-1, neuropilin-1; PPARg, peroxisome proliferator-activated receptor 2; ssRNA, synthetic standard RNA.

To confirm that FLFCs and primary adipocytes were similar in terms of their potential role in hematopoiesis, we cotransplanted FLFCs and CD34⁺ progenitors under the kidney capsules of 16 NOD/SCID mice. In these conditions, FLFCs recovered their adipocyte morphology (Fig. 2A) and were stained positively with anti-human adiponectin antibody (Fig. 2C), whereas cells derived from CD34⁺ progenitors were positive for Mac-387 (a specific human macrophage marker) (Fig. 2D) and negative for chloroacetate esterase activity (Fig. 2F). These morphological features were similar to those observed in human femoral BM. Taken together, these findings establish FLFCs as a reliable model for investigating the effect of adipocytes on myelo-monocytic differentiation, as well as their mechanism of action.

View larger version (100K): [in this window] [in a new window]   Figure 2. Histological analysis of a graft of FLFC and CD34+ under the kidney capsule of NOD/SCID mice shows the differentiation of CD34+ into macrophages. (A): Hematoxylin and eosin stain showed the morphology of the graft (magnification, x10). (C): A goat monoclonal anti-human adip antibody identified human adipocytes (x40). (B): Negative control with NGS (x40). (D): Human macrophages were revealed with a monoclonal anti-human Mac-387 antibody (x40). (E): CEA identified granulopoiesis in human bone marrow (x100). (F): The absence of CEA in the graft excluded the presence of granulopoiesis (x40). Results are representative of three independent experiments. Abbreviations: adip, adiponectin; CEA, chloroacetate esterase activity; FLFC, fibroblast-like fat cells; NGS, normal goat serum.

View larger version (55K): [in this window] [in a new window]   Figure 3. Cell-to-cell contact impaired granulopoiesis through the inhibition of G-CSF production. (A): Mo, PreGra, and segmented PNN were identified by May-Grünwald-Giemsa (MGG) FLFC/CD34+ C and T (magnification, x10 and x50). (B): G-CSF and GM-CSF mRNA expression was analyzed by reverse transcription (RT)-polymerase chain reaction (PCR) in FLFC, SF, and MDSC cultured alone for 1 W (lanes 1, 5, and 9, respectively) and 5 W (lanes 2, 6, and 10, respectively) or cocultured with CD34+ for 1 W (lanes 3, 7, and 11, respectively) and 5 W (lanes 4, 8, and 12, respectively). GAPDH (housekeeping gene) and ssRNA were used to validate the RT-PCR. (C): The presence of G-CSF in the supernatant of FLFC/CD34+ in T, C, and F was analyzed by enzyme-linked immunosorbent assay after 1, 3, and 5 W of culture. (D): Mo and PNN were identified by MGG staining of FLFC/CD34+ C with or without G-CSF (magnification, x100). Data shown in each panels are representative of three independent experiments. Abbreviations: C, cocultures in cell-to-cell contact conditions; F, cultures of fibroblast-like fat cells alone; FLFC, fibroblast-like fat cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MDSC, marrow-derived stromal cells; Mo, macrophages; PNN, polynuclear neutrophil; PreGra, granulocyte precursors; Sd, standard deviation; SF, skin fibroblasts; ssRNA, synthetic standard RNA; T, cocultures in transwell; und, undetectable; W, week.

View larger version (101K): [in this window] [in a new window]   Figure 4. FLFCs impaired the production of G-CSF by Mo and inhibited pregranulocyte differentiation induced by Mo by a mechanism dependent on cell-to-cell contact. (A): G-CSF was detected by immunostaining of FLFCs/CD34+ cocultures in transwell conditions (Ab, Ac) (original magnification, x100); negative control used only the second-step antiserum (Aa) (original magnification, x50). (B): PNN differentiation was evaluated morphologically on cytospins stained with May-Grünwald-Giemsa. (Ba): Immature granulocyte precursors obtained by stimulation of CD34+ progenitors with stem cell factor (50 ng/ml), interleukin-3 (10 ng/ml), granulocyte-macrophage colony-stimulating factor (5 ng/ml), and G-CSF (10 ng/ml) for 7 days. (Bb): Coculture of immature granulocyte precursors with Mo after 9 days (day 16 from the beginning of experiments) in the absence of exogenous cytokines. (Bc): Coculture of immature granulocytes precursors with Mo and FLFC after 9 days. (C): The absolute number of PNN was evaluated in pregranulocyte/Mo cocultures in the absence or in the presence of FLFC. (Results are expressed as mean ± SD from three independent experiments; p < .05.) (D): G-CSF expression detected by immunostaining of adherent cell from Mo and pregranulocyte cocultures with or without FLFCs. (Da): Negative control. (Db, Dc): Positive staining on cocultures of Mo and pregranulocytes without (Db) and with (Dc) FLFCs (original magnification, x40). Abbreviations: F, cultures of fibroblast-like fat cells alone, FLFC, fibroblast-like fat cells; G-CSF, granulocyte-colony stimulating factor; Mo, macrophages; PNN, polynuclear neutrophils; PreGra, granulocyte precursors.

View larger version (40K): [in this window] [in a new window]   Figure 5. Neutralization of NP-1 induced G-CSF production in FLFC/CD34+ and FLFC/Mo cocultures and induced PNN differentiation in FLFC/CD34 and FLFC/PreGra cocultures. (A): NP-1 mRNA expression increased in FLFC/CD34+ cocultures. mRNA levels are expressed in A.U as a ratio between NP-1 polymerase chain reaction product intensity and 28S housekeeping gene after revelation on polyacrylamide gel and quantification by PhosphorImager (GE Healthcare, Little Chalfont, U.K., http://www.gehealthcare.com) and Molecular Analyst software (Bio-Rad, Hercules, CA, http://www.bio-rad.com). (B): NP-1 expression was analyzed by flow cytometry after immunostaining in FLFC cultured alone or with CD34+ cells. Values represent the mean percentage of all assessed cells positively stained and the MFI. (C): Mo and PNN were identified by May-Grünwald-Giemsa (MGG) staining of FLFCs/CD34+ cocultures in the absence (control) and in the presence of neutralizing NP-1 antibody. (D): The production of G-CSF was assessed by enzyme-linked immunosorbent assay (ELISA) in the same culture conditions (the results are expressed as mean ± SD from three independent experiments). (E): Mo, PreGra, and PNN were identified by MGG staining in cocultures of FLFCs, Mo, and PreGra without (control) or with anti-NP-1 neutralizing antibody (magnification, x50). (F): The absolute number of PNN was evaluated in the same culture conditions (results are expressed as mean from three independent experiments; p < .05). (G): The production of G-CSF was assessed by ELISA in the same culture conditions. Abbreviations: A.U, arbitrary units; F, cultures of fibroblast-like fat cells alone, FLFC, fibroblast-like fat cells; G-CSF, granulocyte-colony stimulating factor; MFI, mean fluorescence intensity; Mo, macrophages; NP-1, neuropilin-1; PNN, polynuclear neutrophil; PreGra, granulocyte precursors; und, undetectable.

IL-1β and Dexamethasone Modulate FLFC Properties to Restore Granulopoiesis
In some inflammatory conditions and during corticosteroid treatment, the number of circulating PNNs is increased in peripheral blood. We set out to investigate the mechanisms of this process, which remain largely unknown, by evaluating whether elevated levels of inflammatory cytokines such as IL-1β and corticosteroids can modulate the properties of adipocytes, namely their capacity to inhibit granulopoiesis. In the first set of experiments, we found that IL-1β stimulated FLFCs to produce G-CSF on both transcriptional and protein levels (Fig. 6Aa, 6Ab). A full PNN maturation occurred when CD34⁺ cells and FLFCs were cocultured with IL-1β in conditions allowing cell-to-cell contact (Fig. 6Ac). Nevertheless, the recovery of G-CSF production in transwell chamber assays did not depend on IL-1β, which was detected in neither FLFC nor FLFC/CD34⁺ cocultures (Fig. 6Ad). In the second set of experiments, we used dexamethasone, which increased intracellular G-CSF expression and downregulated NP-1 expression in FLFC and Mo cocultures (Fig. 6B). These experiments support the notion that BM adipocytes may contribute to the control of granulopoiesis in pathological situations, suggesting that increased PNN counts after corticosteroid therapy may be, at least in part, the consequence of NP-1 downregulation.

View larger version (39K): [in this window] [in a new window]   Figure 6. IL-1β and DM modulate FLFC properties. (Aa): G-CSF expression analyzed by reverse transcription (RT)-polymerase chain reaction (PCR) in FLFCs stimulated (+) or not (–) by IL-1β. Samples were analyzed in duplicate. 28S (housekeeping gene) and ssRNA were used to validate the RT-PCR. (Ab): G-CSF production was assessed by enzyme-linked immunosorbent assay (ELISA) in the same culture conditions (results are expressed as mean ± SD of three samples). (Ac): Mo and PNN were identified by May-Grünwald-Giemsa in cell-to-cell contact conditions in the absence and in the presence of IL-1β (magnification, x10 and x50, as indicated). (Ad): IL-1β production was assessed by ELISA in FLFC/CD34+ C, T, and F alone; C + IL-1β represents a positive control (IL-1β was added in an FLFC/CD34+ coculture). (B): Intracellular G-CSF and NP-1 expression was analyzed by flow cytometry after immunostaining of FLFC/Mo cocultures treated with 0.1 µM DM. Values represent the mean percentage of all assessed cells positively stained and the mean fluorescence intensity. Abbreviations: C, cocultures in cell-to-cell contact conditions; DM, dexamethasone; F, cultures of fibroblast-like fat cells alone, FLFC, fibroblast-like fat cells; G-CSF, granulocyte-colony stimulating factor; IL, interleukin; Mo, macrophages; NP-1, neuropilin-1; PNN, polynuclear neutrophil; T, cocultures in transwell; und, undetectable.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

Before setting out on this study based on the use of FLFCs derived from adipocytes isolated from the bone marrow, we made sure of their physiological relevance according to phenotypical and functional criteria. First, we established that FLFCs express the majority of intermediate, late, and very late markers of adipogenic differentiation [41, 42], such as LPL [43, 44], PPAR2 [45–47], G3PDH [48], aP2 [49], CD36 [50, 51], adiponectin [52], and leptin [53, 54]. Second, we assessed the expression of Pref-1/dlk-1 by FLFCs, a marker shared with primary adipocytes, which is not displayed by in vitro-differentiated adipocytes from 3T3 cells [34], and other stromal cell lines, including MSC and HS5. Third, we confirmed that FLFCs expressed NP-1, as previously reported for femoral BM adipocytes [18]. Finally and most importantly, we found that FLFCs adopted the morphology of adipocytes in vivo and supported the differentiation of CD34⁺ progenitor cells into Mos. For all these reasons, we consider FLFCs a relevant model allowing the investigation of the influence of adipocytes on hematopoietic cell differentiation in vitro.

Cocultures of CD34⁺ progenitor cells and FLFCs set up in cell-to-cell contact or transwell conditions gave rise to Mos. FLFCs constitutively produced cytokines involved in Mo differentiation, such as GM-CSF and M-CSF (data not shown). This pattern of cytokine production is a general property of adipocytes regardless of their location, since it has also been described in adipocytes from mammary tissue [52, 55] and in BM-MSC [56].

Transwell chamber experiments showed that contact between FLFCs and CD34⁺ was not critical for Mo differentiation but prevented the generation of mature granulocytes and the production G-CSF. This observation was in agreement with our previous data on the MS5 cell line, which lost its capacity to sustain granulopoiesis after differentiation into adipocytes [31]. GM-CSF was constitutively expressed by FLFCs but increased in cocultures with CD34⁺ progenitors, which is probably the reason why a few immature granulocytes differentiate in culture [57, 58]. This growth factor has also been demonstrated in freshly isolated BM adipocytes (data not shown), as well as in BM preadipocytes [59]. By contrast, G-CSF was not detected in cocultures with cell-to-cell contact, but it restored granulopoiesis when added exogenously. It has been reported previously that adipocytes differentiated in vitro either from bone marrow MSC [60] or from subcutaneous adipose tissue stroma-vascular fraction cells (SVF) [61] support the generation of mature granulocytes from immature precursors, even though G-CSF production was assessed only in SVF cultures. According to another study, G-CSF production by MSC was downregulated after adipocyte differentiation [56]. These apparent discrepancies may be explained by the distinct cellular origins [62] and/or culture conditions in which adipocytes are differentiated.

To our knowledge, this is the first study using primary BM adipocytes in coculture with CD34⁺ to assess the effect of adipocytes on hematopoiesis and the inhibition of G-CSF production through cell-to-cell contact. We postulated that NP-1 could be implicated in this inhibition because (a) NP-1 is differentially expressed in hematopoietic iliac crest BM (rich in HCs and NP-1^low) and femoral BM (poor in HCs and NP-1^high) [18]; (b) the expression of NP-1 increased in FLFCs and CD34⁺ cocultures; (c) the role of NP-1 in the modulation of cytokine production, including TPO and Flt-3 ligand, by stromal cells has been reported before [25]; and (d) NP-1 plays a role in various cell-to-cell contact conditions [23].

Blocking NP1 by its specific antibody could indeed restore G-CSF production and granulopoiesis. Interestingly, mature PNNs counts were not increased in either Mo/PreGra or in FLFCs/PreGra cocultures after NP-1 inhibition (data not shown).

This observation led us to conclude that NP-1 is involved in interactions between macrophages and FLFCs. It has already been reported that NP-1 participated in the interactions between the murine MS5 cell line and primitive hematopoietic cells [25]. However, the mechanisms accounting for the inhibition of G-CSF production by Mos by NP1 in the presence of FLFCs remained obscure. The lack of expression of vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2 in FLFCs and Mo in this system of culture argues against a role of VEGF (data not shown). Sema 3A, another ligand of NP-1, was expressed in all culture conditions (FLFCs and MDSCs alone or cocultured with CD34⁺), ruling out its implication in the inhibition of G-CSF production. Therefore, as suggested by other investigators, NP-1 may target other signaling receptors or establish homotypic interactions [63–65].

	CONCLUSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Our results support the notion that adipocytes are active members of the BM microenvironment playing a critical role in the regulation of hematopoiesis. It is tempting to speculate that they regulate the balance between the production of Mo and granulocytes, facilitating increased production of granulocytes in pathological conditions. Therefore, a deregulation of adipocytes functions and/or numbers may result in hematological, inflammatory, or immunological disorders. In agreement with this hypothesis, IL-1, a proinflammatory cytokine, induces G-CSF production and PNN maturation in cocultures between CD34⁺ progenitors and FLFCs in cell-to-cell contact conditions. Thus, the increased granulocyte counts in some inflammatory diseases could be explained by an effect of IL-1 on the adipocyte function. Furthermore, dexamethasone, a corticosteroid known to induce increased polynuclear neutrophil numbers, downregulates NP-1 in FLFC/Mo cocultures similarly to what has already been reported in MS5 cell line, and this downregulation could account for the elevated PNN numbers in patients treated with dexamethasone.

Targeting BM adipocytes could be useful in some BM failure syndromes in which adipocytes should no longer be viewed as cells occupying the space left empty by hematopoietic cells but as active elements in the physiopathological process. Further work, however, is necessary to understand the molecular mechanisms involved in NP-1-induced inhibition of G-CSF production by Mo, which may help to define new therapeutic strategies in some neutropenia of undetermined origin.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
This work was supported by the Fond de la Recherche Scientifique-Fonds National de la Recherche Scientifique (FRS-FNRS), the Fédération Belge Contre le Cancer, Cancéropole, la Ligue Nationale contre le Cancer, Association pour la Recherche sur le Cancer, Institut National du Cancer, la Fondation pour la Recherche Médicale, la Fondation de France, and the Commissariat Général aux Relations Internationales (Tournesol). Z.B.-C., G.P., and A.Be. are Télévie (FNRS) fellows. Y.L. is supported by a grant from la Ligue Nationale contre le Cancer. Excellent technical assistance was provided by M.J. Nix, D. Delneuville, A. Heyeres, and E. Frantzen. We thank Dr. A. Rodrigues and P. Gillet for performing the surgery and providing biopsies. We thank also J. Cadranel, C.M. Lapière, Dr. A. Colige, Dr. A. Kolodkin, and Dr. D. Ginty (Johns Hopkins University) for gifts and expertise. Z.B.-C. and Y.L. contributed equally to this work. M.P.D., O.H., and B.V.N. codirected this work.

	FOOTNOTES

 
Author contributions: Z.B.-C. and Y.L.: collection and assembly of data, conception and design, data analysis and interpretation, manuscript writing; G.P.: collection and assembly of data, data analysis and interpretation; A.T. and A.Br.: provision of study material or patients; C.H.: data analysis and interpretation, administrative support; M.M. and M.L.A.: data analysis and interpretation; A.Be., P.M., C.L., D.M.-D.-C., and V.A.: collection and assembly of data; E.S.: manuscript writing; M.D. and J.B.: final approval of manuscript; B.V.N., O.H., and M.P.D.: conception and design, data analysis and interpretation, manuscript writing, financial support.

	REFERENCES

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 

1. Bianco P, Riminucci M, Gronthos S et al. Bone marrow stromal stem cells: Nature, biology, and potential applications. STEM CELLS 2001;19:180–192.[Abstract/Free Full Text]

2. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993;81:2844–2853.[Abstract/Free Full Text]

3. Heyworth CM, Whetton AD, Nicholls S et al. Stem cell factor directly stimulates the development of enriched granulocyte-macrophage colony-forming cells and promotes the effects of other colony-stimulating factors. Blood 1992;80:2230–2236.[Abstract/Free Full Text]

4. Aglietta M, Pasquino P, Sanavio F et al. Granulocyte-macrophage colony stimulating factor and interleukin 3: Target cells and kinetics of response in vivo. STEM CELLS 1993;11(suppl 2):83–87.

5. Jacobsen K, Kravitz J, Kincade PW et al. Adhesion receptors on bone marrow stromal cells: In vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice. Blood 1996;87:73–82.[Abstract/Free Full Text]

6. Metcalf D. The molecular control of proliferation and differentiation in hemopoietic cells. C R Acad Sci III 1993;316:860–870.[Medline]

7. Nakamura K, Kosaka M, Mizuguchi T et al. Effect of erythroid differentiation factor on maintenance of human hematopoietic cells in co-cultures with allogenic stromal cells. Biochem Biophys Res Commun 1993;194:1103–1110.[CrossRef][Medline]

8. Rafii S, Avecilla S, Shmelkov S et al. Angiogenic factors reconstitute hematopoiesis by recruiting stem cells from bone marrow microenvironment. Ann N Y Acad Sci 2003;996:49–60.[CrossRef][Medline]

9. Kume K, Satomura K, Nishisho S et al. Potential role of leptin in endochondral ossification. J Histochem Cytochem 2002;50:159–169.[Abstract/Free Full Text]

10. Hattori H, Ishihara M, Fukuda T et al. Establishment of a novel method for enriching osteoblast progenitors from adipose tissues using a difference in cell adhesive properties. Biochem Biophys Res Commun 2006;343:1118–1123.[CrossRef][Medline]

11. Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 2004;89:2548–2556.[Abstract/Free Full Text]

12. Gregoire FM. Adipocyte differentiation: From fibroblast to endocrine cell. Exp Biol Med (Maywood) 2001;226:997–1002.[Abstract/Free Full Text]

13. Baratta M. Leptin—From a signal of adiposity to a hormonal mediator in peripheral tissues. Med Sci Monit 2002;8:RA282–RA292.[Medline]

14. Fukumura D, Ushiyama A, Duda DG et al. Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ Res 2003;93:e88–e97.[CrossRef][Medline]

15. Hattori K, Sumi T, Yasui T et al. VEGF mRNA in adipocytes increase with rebound weight-gain after diet-restriction. Int J Mol Med 2004;13:395–399.[Medline]

16. Frühbeck G, Gomez-Ambrosi J. Modulation of the leptin-induced white adipose tissue lipolysis by nitric oxide. Cell Signal 2001;13:827–833.[CrossRef][Medline]

17. Mohamed-Ali V, Goodrick S, Rawesh A et al. Subcutaneous adipose tissue releases interleukin-6, but not tumor necrosis factor-alpha, in vivo. J Clin Endocrinol Metab 1997;82:4196–4200.[Abstract/Free Full Text]

18. Belaid Z, Hubint F, Humblet C et al. Differential expression of vascular endothelial growth factor and its receptors in hematopoietic and fatty bone marrow: Evidence that neuropilin-1 is produced by fat cells. Haematologica 2005;90:400–401.[Abstract/Free Full Text]

19. He Z, Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell 1997;90:739–751.[CrossRef][Medline]

20. Soker S, Takashima S, Miao HQ et al. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998;92:735–745.[CrossRef][Medline]

21. Kolodkin AL, Levengood DV, Rowe EG et al. Neuropilin is a semaphorin III receptor. Cell 1997;90:753–762.[CrossRef][Medline]

22. Neufeld G, Cohen T, Shraga N et al. The neuropilins: Multifunctional semaphorin and VEGF receptors that modulate axon guidance and angiogenesis. Trends Cardiovasc Med 2002;12:13–19.[CrossRef][Medline]

23. Tordjman R, Lepelletier Y, Lemarchandel V et al. A neuronal receptor, neuropilin-1, is essential for the initiation of the primary immune response. Nat Immunol 2002;3:477–482.[Medline]

24. Lepelletier Y, Smaniotto S, Hadj-Slimane R et al. Control of human thymocyte migration by Neuropilin-1/Semaphorin-3A-mediated interactions. Proc Natl Acad Sci U S A 2007;104:5545–5550.[Abstract/Free Full Text]

25. Tordjman R, Ortega N, Coulombel L et al. Neuropilin-1 is expressed on bone marrow stromal cells: A novel interaction with hematopoietic cells? Blood 1999;94:2301–2309.[Abstract/Free Full Text]

26. Lieschke GJ, Grail D, Hodgson G et al. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 1994;84:1737–1746.[Abstract/Free Full Text]

27. Liu F, Wu HY, Wesselschmidt R et al. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice. Immunity 1996;5:491–501.[CrossRef][Medline]

28. Nicola NA, Metcalf D, Johnson GR et al. Separation of functionally distinct human granulocyte-macrophage colony-stimulating factors. Blood 1979;54:614–627.[Abstract/Free Full Text]

29. Sugihara H, Yonemitsu N, Miyabara S et al. Proliferation of unilocular fat cells in the primary culture. J Lipid Res 1987;28:1038–1045.[Abstract]

30. Sugihara H, Yonemitsu N, Miyabara S et al. Primary cultures of unilocular fat cells: Characteristics of growth in vitro and changes in differentiation properties. Differentiation 1986;31:42–49.[CrossRef][Medline]

31. Hubin F, Humblet C, Belaid Z et al. Murine bone marrow stromal cells sustain in vivo the survival of hematopoietic stem cells and the granulopoietic differentiation of more mature progenitors. STEM CELLS 2005;23:1626–1633.[Abstract/Free Full Text]

32. Oh H, Takagi H, Otani A et al. Selective induction of neuropilin-1 by vascular endothelial growth factor (VEGF): A mechanism contributing to VEGF-induced angiogenesis. Proc Natl Acad Sci U S A 2002;99:383–388.[Abstract/Free Full Text]

33. Matthies AM, Low QE, Lingen MW et al. Neuropilin-1 participates in wound angiogenesis. Am J Pathol 2002;160:289–296.[Abstract/Free Full Text]

34. Smas CM, Sul HS. Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell 1993;73:725–734.[CrossRef][Medline]

35. Gimble JM, Robinson CE, Wu X et al. The function of adipocytes in the bone marrow stroma: An update. Bone 1996;19:421–428.[Medline]

36. Tavassoli M, Crosby WH. Bone marrow histogenesis: A comparison of fatty and red marrow. Science 1970;169:291–293.[Abstract/Free Full Text]

37. Tavassoli M. In Tavassoli M, ed. Handbook of the Hemopoietic Microenvironment. Fatty involution of marrow and the role of adipose tissue in hemopoiesis. Clifton, NJ: Humana Press, 1989:157-187.

38. Cancello R, Henegar C, Viguerie N et al. Reduction of macrophage infiltration and chemoattractant gene expression changes in white adipose tissue of morbidly obese subjects after surgery-induced weight loss. Diabetes 2005;54:2277–2286.[Abstract/Free Full Text]

39. Kanda H, Tateya S, Tamori Y et al. MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest 2006;116:1494–1505.[CrossRef][Medline]

40. Bornstein SR, Abu-Asab M, Glasow A et al. Immunohistochemical and ultrastructural localization of leptin and leptin receptor in human white adipose tissue and differentiating human adipose cells in primary culture. Diabetes 2000;49:532–538.[Abstract]

41. Urs S, Smith C, Campbell B et al. Gene expression profiling in human preadipocytes and adipocytes by microarray analysis. J Nutr 2004;134:762–770.[Abstract/Free Full Text]

42. Hung SC, Chang CF, Ma HL et al. Gene expression profiles of early adipogenesis in human mesenchymal stem cells. Gene 2004;340:141–150.[CrossRef][Medline]

43. Ruge T, Sukonina V, Myrnas T et al. Lipoprotein lipase activity/mass ratio is higher in omental than in subcutaneous adipose tissue. Eur J Clin Invest 2006;36:16–21.[Medline]

44. Schilling T, Noth U, Klein-Hitpass L et al. Plasticity in adipogenesis and osteogenesis of human mesenchymal stem cells. Mol Cell Endocrinol 2007;271:1–17.[CrossRef][Medline]

45. Spiegelman BM, Hu E, Kim JB et al. PPAR gamma and the control of adipogenesis. Biochimie 1997;79:111–112.[Medline]

46. Gurnell M. Peroxisome proliferator-activated receptor gamma and the regulation of adipocyte function: Lessons from human genetic studies. Best Pract Res Clin Endocrinol Metab 2005;19:501–523.[CrossRef][Medline]

47. Hummasti S, Tontonoz P. The peroxisome proliferator-activated receptor N-terminal domain controls isotype-selective gene expression and adipogenesis. Mol Endocrinol 2006;20:1261–1275.[Abstract/Free Full Text]

48. MacDougald OA, Lane MD. Transcriptional regulation of gene expression during adipocyte differentiation. Ann Rev Biochem 1995;64:345–373.[CrossRef][Medline]

49. Mackay DL, Tesar PJ, Liang LN et al. Characterizing medullary and human mesenchymal stem cell-derived adipocytes. J Cell Physiol 2006;207:722–728.[CrossRef][Medline]

50. Ibrahimi A, Abumrad NA. Role of CD36 in membrane transport of long-chain fatty acids. Curr Opin Clin Nutr Metab Care 2002;5:139–145.[CrossRef][Medline]

51. Rodrigue-Way A, Demers A, Ong H et al. A growth hormone-releasing peptide promotes mitochondrial biogenesis and a fat burning-like phenotype through scavenger receptor CD36 in white adipocytes. Endocrinology 2007;148:1009–1018.[Abstract/Free Full Text]

52. Sell H, Dietze-Schroeder D, Eckardt K et al. Cytokine secretion by human adipocytes is differentially regulated by adiponectin, AICAR, and troglitazone. Biochem Biophys Res Commun 2006;343:700–706.[Medline]

53. Hwang CS, Loftus TM, Mandrup S et al. Adipocyte differentiation and leptin expression. Annual review of cell and developmental biology 1997;13:231–259.[CrossRef][Medline]

54. Zhang F, Chen Y, Heiman M et al. Leptin: Structure, function and biology. Vitam Horm 2005;71:345–372.[CrossRef][Medline]

55. Levine JA, Jensen MD, Eberhardt NL et al. Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth. J Clin Invest 1998;101:1557–1564.[Medline]

56. Kim DH, Yoo KH, Choi KS et al. Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell. Cytokine 2005;31:119–126.[CrossRef][Medline]

57. Bonnier S, Campos L, Froehlich C et al. Modification of in vitro hematopoiesis induced by addition of GM-CSF in long term normal human bone marrow cultures [in French]. Pathol Biol (Paris) 1991;39:271–276.[Medline]

58. Lee MY, Fukunaga R, Lee TJ et al. Bone modulation in sustained hematopoietic stimulation in mice. Blood 1991;77:2135–2141.[Abstract/Free Full Text]

59. Aoki S, Toda S, Ando T et al. Bone marrow stromal cells, preadipocytes, and dermal fibroblasts promote epidermal regeneration in their distinctive fashions. Mol Biol Cell 2004;15:4647–4657.[Abstract/Free Full Text]

60. Corre J, Planat-Benard V, Corberand JX et al. Human bone marrow adipocytes support complete myeloid and lymphoid differentiation from human CD34 cells. Br J Haematol 2004;127:344–347.[CrossRef][Medline]

61. Corre J, Barreau C, Cousin B et al. Human subcutaneous adipose cells support complete differentiation but not self-renewal of hematopoietic progenitors. J Cell Physiol 2006;208:282–288.[CrossRef][Medline]

62. Zhou X, Li D, Yin J et al. CLA differently regulates adipogenesis in stromal vascular cells from porcine subcutaneous adipose and skeletal muscle. J Lipid Res 2007;48:1701–1709.[Abstract/Free Full Text]

63. Bachelder RE, Crago A, Chung J et al. Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res 2001;61:5736–5740.[Abstract/Free Full Text]

64. Bachelder RE, Lipscomb EA, Lin X et al. Competing autocrine pathways involving alternative neuropilin-1 ligands regulate chemotaxis of carcinoma cells. Cancer Res 2003;63:5230–5233.[Abstract/Free Full Text]

65. Murga M, Fernandez-Capetillo O, Tosato G. Neuropilin-1 regulates attachment in human endothelial cells independently of vascular endothelial growth factor receptor-2. Blood 2005;105:1992–1999.[Abstract/Free Full Text]

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