Published Ahead of Print on July 16, 2012, as doi:10.3324/haematol.2012.065201. Copyright 2012 Ferrata Storti Foundation.
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Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells by Sabine Stopp, Martin Bornhäuser, Fernando Ugarte, Manja Wobus, Matthias Kuhn, Sebastian Brenner, and Sebastian Thieme Haematologica 2012 [Epub ahead of print] Citation: Stopp S, Bornhäuser M, Ugarte F, Wobus M, Kuhn M, Brenner S, and Thieme S. Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells. Haematologica. 2012; 97:xxx doi:10.3324/haematol.2012.065201 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Haematologica (pISSN: 0390-6078, eISSN: 1592-8721, NLM ID: 0417435, www.haematologica.org) publishes peer-reviewed papers across all areas of experimental and clinical hematology. The journal is owned by the Ferrata Storti Foundation, a non-profit organization, and serves the scientific community with strict adherence to the principles of open access publishing (www.doaj.org). In addition, the journal makes every paper published immediately available in PubMed Central (PMC), the US National Institutes of Health (NIH) free digital archive of biomedical and life sciences journal literature.
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DOI: 10.3324/haematol.2012.065201
Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells
Sabine Stopp1, Martin Bornhäuser1,3, Fernando Ugarte2, Manja Wobus1, Matthias Kuhn4, Sebastian Brenner,2,3# and Sebastian Thieme2# 1
Medical Clinic and Policlinic I; 2Department of Pediatrics; University Hospital “Carl
Gustav Carus” Dresden; 3Center for Regenerative Therapies Dresden, and 4Institute for Medical Informatics and Biometry, Technical University Dresden #SB and ST contributed equally to this manuscript
Correspondence Sebastian Thieme, Department of Pediatrics, University Hospital “Carl Gustav Carus”, Fetscherstraße 74, 01309 Dresden. Phone: international +49.351.4586885. Fax: international +49.351.4586333. E-mail
[email protected]
Acknowledgments The authors would like to thank Ms. Katrin Müller for isolation and expansion of primary human MSC and Mrs. Katrin Navratiel for isolation of CD34+ HSPC from cord blood. Funding This work was supported by the Collaborative Research Grants SFB-655 and FOR1586 from the German Research Foundation (DFG, SFB-655 project B6 to SB, project B2 to MB, Project 1 of FOR 1586 ‘Skelmet’ to MW and MB) and the Center for Regenerative
Therapies,
Dresden. 1
DOI: 10.3324/haematol.2012.065201
Abstract Background The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo.(1) The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. Design and Methods We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Results Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Conclusions
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Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell–cell contact.
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Introduction Multipotent human mesenchymal stromal cells (hMSC) support the growth of hematopoietic stem and progenitor cells (HSPC) during ex vivo co-culture.(2-4) hMSC produce various growth factors, adhesion molecules, and matrix proteins contributing to the formation of stem cell niches, thereby controlling the homing, maintenance, and differentiation of HSPC.(2;5) Well-studied signaling pathways within this niche include Notch, Wnt, and Hedgehog.(6;7) Soluble signaling molecules include cytokines, growth factors, or chemokines (e.g., stem cell factor (SCF), the FLT3 ligand (FLT3-L), and stromal derived factor-1 (SDF-1)).(8;9) It is believed that direct cell–cell contact mediated by adhesion molecules is essential for the maintenance of immature HSPC. Several adhesion molecules (VCAM-1, ICAM-1, N-cadherin, and NCAM) are known to be important for niche formation(5;7-9) and hematopoiesis. The melanoma cell adhesion molecule (MCAM/CD146) is used as a marker for mesenchymal stromal cells. In a xenotransplantation model, Sacchetti et al. demonstrated that culture-expanded MCAM+ bone marrow stromal cells reconstituted the hematopoietic microenvironment.(1) Furthermore, the expression pattern of MCAM on bone marrow-derived mesenchymal stromal cells correlated with their in situ localization.(10) However, the exact function of MCAM within the human bone marrow niche is unclear. MCAM is a 113 kDa glycoprotein that belongs to the immunoglobulin (Ig) superfamily of cell adhesion molecules. It contains an extracellular domain with five Ig domains (V-V-C2-C2-C2), a transmembrane domain, and a cytoplasmic domain with potential recognition sequences for protein kinases.(11) MCAM orthologs have been identified in the mouse, rat, chicken, and zebrafish.(12) Human MCAM was originally identified as a marker for melanoma progression and metastasis. MCAM is further expressed by the vascular endothelium, smooth muscle cells, activated T lymphocytes, and
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bone marrow stromal cells.(11) MCAM function has been extensively studied in melanomas and other types of cancer (prostate cancer and breast cancer), but the ligand for MCAM has not yet been identified.(12-14) This study aimed to elucidate the impact of MCAM expression on the functional properties of hMSC and the maintenance of HSPC in co-culture. Thus, we generated primary hMSC that stably expressed shRNA against MCAM or that overexpressed an MCAM coding sequence (CDS) through lentiviral vector gene transfer. Our findings indicate that MCAM expression has a pivotal role in hMSC differentiation and the maintenance of HSPC through direct cell–cell contact.
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Design and Methods Isolation of hMSC and HSPC Primary hMSC and HSPC were isolated from healthy donors after informed consent and the approval of the local ethics committee. Primary hMSC were isolated from bone marrow aspirates, as described previously.(15) CD34+ HSPC were purified from either mobilized peripheral blood or umbilical cord blood using CD34 antibodyconjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The purity of hMSC and HSPC cells were evaluated by flow cytometry (hMSC: CD45−, CD34−, CD73+, CD90+, CD105+, CD166+; HSPC: CD45+, CD34+, CD133+, CD38-/dim). hMSCs were cultured in DMEM GlutaMax (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FCS; Biochrom, Cambridge, UK). All experiments were performed with hMSC that were passaged only once to avoid any variations due to the aging of cells. Isolated HSPCs were cultured in corresponding media.
Lentiviral vectors, virus vector production, and hMSC transduction Plasmid pLKO.1 vectors encoding shRNAs that targeted human MCAM (KD1 and KD2) were obtained from OpenBiosystems (Huntsville, AL, USA). Control pLKO.1 vectors (empty vector control or vector with shRNA targeting eGFP) were obtained from Sigma-Aldrich (Taufkirchen, Germany). The MCAM coding sequence was amplified from a cDNA library and directionally cloned into the lentiviral vector pRRL.SIN.cPPT.SFFV.GFP.WPRE(16;17) (kindly provided by Christopher Baum, Hannover, Germany) fused with an IRES-GFP sequence (MCAM-GFP) using XhoI/BamHI restriction sites. To produce lentiviral vector particles, HEK293T cells were transfected with lentiviral vectors in combination with the packaging plasmids psPAX and pVSVg using PEI.(18) Lentivirus-vector containing media were collected
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48 h after transfection. Primary hMSC were infected thrice with lentiviral vector particles (0.5× viral supernatant) in the presence of 1 µg mL−1 protamine every 12 h.
Assessment of cell differentiation, proliferation, β-galactosidase staining and migration of transduced hMSC Twelve hours after seeding the transduced hMSC, the medium was replaced with differentiation medium. Osteogenic differentiation medium was supplemented with 10 mM β-glycerophosphate, 5 µ M dexamethasone, and 200 µ M ascorbic acid (SigmaAldrich). Mineralization was analyzed with von Kossa staining 10 days after differentiation. In brief, cells were washed once with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde, washed, stained with 1% AgNO3, and developed using 0.1% pyrogallol. Alkaline phosphatase activity was measured in cell extracts using the pNPP Liquid Substrate System (Sigma-Aldrich). The adipogenic differentiation medium contained 50 µ M dexamethasone, 500 µM 3-isobutyl-1methylxanthine, 1 mg mL−1 insulin, and 100 µ M indomethacin (Sigma-Aldrich). Adipocytes were quantified after 14 days using Oil Red staining. In brief, cells were washed with PBS, fixed with 4% paraformaldehyde, washed, and stained with Oil Red solution for 15 min at room temperature. Excess stain was removed using two washes with 60% ethanol. Adipocytes were visualized using a microscope (Zeiss Axiovert 400M; magnification ×100). Cell proliferation was measured using a 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Roche, Mannheim, Germany) assay. In brief, 1000 cells/96-wells were seeded in five replicates and grown for 96 h. MTT was added to prepare a final concentration of 0.5 mg mL−1. Formazan crystals were solubilized overnight and the absorbance was measured at 570 nm. For the EdU (5-ethynyl-2’-deoxyuridine) incorporation assay (Invitrogen), 1.5 × 105 cells were seeded in a T25 flask. Twelve hours after seeding, EdU was added
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to the cell culture medium to prepare a final concentration of 10 µM. After 24 h incubation, cells were harvested and analyzed by flow cytometry, according to the manufacturer’s instructions. Cells without EdU incorporation were used as negative control. Cell cycle analysis was measured simultaneously with EdU incorporation by 7-AAD-staining. To detect senescent cells, 1 × 104 cells/24-well were seeded in duplicate for βgalactosidase staining (Cell Signaling Technology, Boston, MA, USA). Twenty-four hours after seeding, cells were subjected to a β-galactosidase assay, according to the manufacturer’s instructions. In vitro wounding assays were performed using six-well-plates to assess cell migration. Confluent hMSC monolayer were wounded with sterile 1000 µ L pipette tips, washed twice with PBS to remove non-adherent cells, and incubated further in complete growth medium. Wounds were imaged at 0 h and after 20 h using phasecontrast microscopy. To mimic constitutive RhoA activation 10 µM Nocodazole (Sigma Aldrich) was added to cell culture for 20 hours. Cell migration was quantified using the ImageJ program (NIH, Bethesda, MD, USA).
Co-culture of HSPC and transduced hMSC Co-culture of HSPC and hMSC was performed as described previously.(19) To establish comparable, confluent monolayers, equal cell numbers (4 × 104 cells cm−2) of transduced hMSC (MCAM-KD, MCAM-GFP, IRES-GFP, pLKO.1) were seeded into appropriate culture vessels. HSPC were suspended in CellGro SCG medium (CellGenix, Freiburg, Germany) supplemented with 10% FCS (Biochrom), 50 ng mL−1 FLT3L, 50 ng mL−1 SCF, and 50 ng mL−1 IL3 (Peprotech Ltd., London, UK). The HSPC suspension was plated at a density of 1 × 104 cells cm−2 on a confluent hMSC
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layer. At fixed time points, cells were harvested and analyzed by flow cytometry. In some studies, the entire co-culture was trypsinized and analyzed, whereas in others, HSPC were collected with regard to their adhesion and migration properties toward hMSC. In brief, the supernatant of the co-culture was aspirated and the cells in the supernatant (non-adherent fraction) were collected. The hMSC layer was washed twice with PBS to collect and remove the remaining non-adherent HSPC. The remaining HSPC on the hMSC layer (phase-bright cells) were collected through additional intensive washing steps using PBS. The hMSC layer with the HSPC beneath (phase-dim cells) was trypsinized. To exclude any side effect of the trypsin treatment, non-adherent cells and phase-bright cells were also incubated with trypsin for 5 min. The proportion of HSPCs within the different cell fractions was referred to the number of CD45 positive cells using flow cytometry. To monitor the cell division of HSPC, cells were labeled with CFSE cell tracker dye (Invitrogen) at a final concentration of 2.5 µ M and subsequently co-cultured with hMSC, as described above. After five days of co-culture, CFSE-labeled HSPC were collected and analyzed by flow cytometry. The number of cell divisions was quantified according to the CFSE signal intensity using the FlowJo software (TreeStar, version 7.6.1). Non-contact co-culture experiments of HSPC and transduced hMSC were performed as previously described.(3) In brief, freshly isolated HSPC were seeded in transwellinserts (0.5-µm pore size, BD Biosciences, Heidelberg, Germany) above a confluent hMSC monolayer and cultured in complete CellGro medium. At fixed time points, cells were harvested and analyzed by flow cytometry. In the adhesion assays, confluent hMSC feeder layers were established in 24-well plates. Freshly isolated HSPC were labeled with Vybrant DiD (Invitrogen), according to the manufacturer’s instructions. Next, 1.5 × 105 labeled HSPC/24-well were
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seeded in duplicate on an hMSC feeder layer in CellGro medium. After 24 h, nonattached cells were removed and counted. Attached cells were monitored using fluorescence microscopy (Zeiss Axiovert 400M; magnification ×100). Transwell migration assays were performed as described previously.(20) Monolayers of primary hMSC were grown on transwell inserts (5-µm pore size, Corning, NY, USA), washed twice and placed in a 24-well plate, containing DMEM supplemented with 10% FCS. Stromal-derived factor (SDF)-1 (200 ng mL−1; R&D Systems, Minneapolis, MN, USA) was added to the lower chamber. Next, 1.5 × 105 HSPC were placed in the upper chamber and incubated for 24 h at 37°C in a 5% CO2-humidified incubator. Trans-migrated cells were counted using flow cytometry. Spontaneous migration without addition of SDF-1 was used as control.
Long-term culture initiating cell (LTC-IC) assay and colony-forming cell assay Lentiviral vector transduced hMSC were seeded as a confluent monolayer in triplicate in 24-well plates. Twenty-four hours after seeding, 1 × 103 HSPC/24-well were seeded onto a hMSC layer in Myelocult H5100 (Stem Cell Technology, Vancouver, British Columbia, Canada) supplemented with 10−6 M hydrocortisone (SigmaAldrich). Plates were incubated at 37°C, 5% CO2 for four weeks with weekly halfmedium exchanges. Cobblestone-area forming cells (CAFC) were evaluated after four weeks of co-culture. LTC-IC assays were trypsinized and the proportions of CD45+ HSPC were quantified by flow cytometry. In total, 7 × 103 cells were replated in triplicate for secondary colony-forming cell assays (CFUs) in MethoCult GF H4435 (Stem Cell Technology) for 14 days. Formation of CFUs was calculated according to the initially seeded proportion of CD45+ HSPC and qualitatively scored using an inverted light microscope according to standard criteria.
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Fluorescence-activated cell sorting, RhoA pull-down, western blotting, quantitative RT-PCR, and ELISA Ex vivo co-cultured HSPC were harvested as described above. Cells were stained with CD34-APC, CD133-PE, CD45-PerCP, CD38-FITC (Miltenyi Biotec), CD13-PE, CD33-PE, CD10-PE-Cy7, CD56-PE-Cy7 (BD Biosciences) for 45 min at 4°C before being analyzed using the LSRII (BD Biosciences) flow cytometer. For RhoA pull-down, 5 x 105 hMSC were seeded in a T75 flask and maintained for 23 days. Cells were starved overnight in serum-free DMEM and then stimulated with 10% FCS for 5 min at 37°C. Immediately after stimulation, cells were washed once with ice-cold TBS and lysed in lysis buffer. For each pull-down, 500 µ g of total cell lysate was processed according to the manufacturer´s instructions (Thermo Scientific, Rockford, IL, USA) and separated with a 12.5% SDS-PAGE gel. To determine protein expression levels by western blotting, cells were washed with PBS and lysed with extraction buffer (M-Per Mammalian Extraction buffer, Thermo Scientific). Next, 20 µ g of protein was loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 1% BSA in 1× TBST at room temperature for 1 h and incubated with human specific primary antibodies (MCAM (R&D Systems); β-actin (Sigma-Aldrich); Cyclin E, phospho-Rb (Ser807/811), CDK2 (Cell Signaling); PCNA (Santa Cruz); Gapdh (Meridian Life Science, Memphis, USA)) at 4°C overnight, followed by incubation with HRPconjugated secondary antibody (anti-mouse IgG-HRP (Thermo Scientific); anti-rabbit IgG-HRP (GE Healthcare, Munich, Germany)) for 1 h at room temperature before they were developed using an ECLPlus Western Blot detection system (Thermo Scientific). Images were acquired with an LAS3000 camera (Fujifilm, Düsseldorf, Germany) and quantified by densiometry using Image J software. Expression levels were normalized to loading control (Gapdh, b-actin or total RhoA).
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Total RNA was isolated using RNeasy columns (Qiagen, Hilden, Germany) and reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. Expression levels of MCAM, p21, p53, and SDF-1 (Supplementary Table S1) were quantified with a TaqMan assay (Applied Biosystems, Foster City, CA, USA). Relative expression was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). For phalloidin staining, 1 x 104 hMSC were seeded onto poly-L-lysine-coated coverslips for 24 h. To induce RhoA activation specific stress fiber formation 10 µM Nocodazole (Sigma Aldrich) was added to cell culture for 5 hours. Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT), followed by washing thrice with PBS. Human MSC were permeabilized with 0.2% Triton X-100 for 5 min, followed by blocking with 1% BSA in PBST for 30 min. Cells were incubated with phalloidin-rhodamine (Invitrogen) for one hour at RT, followed by three washes with PBS. Nuclei were labeled with 2 µg/ml DAPI for 5 min. Then, coverslips were rinsed once with PBS and mounted in mounting medium. Labeled cells were imaged using Zeiss Axiovert 400M (magnification x400). Human SDF-1 in cell culture supernatants of transduced hMSC was quantified with a DuoSet ELISA Development kit (R&D Systems) according to the manufacturer’s instructions.
Statistical analysis Statistical analysis was performed using paired Student t-tests. To assess qualitative differences in the formation of secondary colonies the Cochran-Mantel-Haenszel test was used. Statistical significance was tested at *p < 0.05; **p < 0.01 and ***p
