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TISSUE-SPECIFIC STEM CELLS

Circulating Bone Marrow-Derived Osteoblast Progenitor Cells Are Recruited to the Bone-Forming Site by the CXCR4/Stromal Cell-Derived Factor-1 Pathway

^aDivision of Gene Therapy Science and
^bDepartment of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

Key Words. Bone marrow cells • Chemokine receptor CXCR4 • Mobilization kinetics • Osteoblast • Peripheral blood Stromal derived factor-1 • Stem/progenitor cell • Tissue regeneration

Correspondence: Correspondence: Katsuto Tamai, M.D., Ph.D., Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Telephone: 81-6-6879-3901; Fax: 81-6-6879-3909; e-mail: tamai{at}gts.med.osaka-u.ac.jp

Received on July 1, 2007; accepted for publication on September 28, 2007.

First published online in STEM CELLS EXPRESS  October 11, 2007.

	ABSTRACT

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

 
Previous studies demonstrated the existence of osteoblastic cells in circulating blood. Recently, we reported that osteoblast progenitor cells (OPCs) in circulation originated from bone marrow and contributed to the formation of ectopic bone induced by implantation of a bone morphogenetic protein (BMP)-2-containing collagen pellet in mouse muscular tissue. However, the character of circulating bone marrow-derived osteoblast progenitor cells (MOPCs) and the precise mechanisms involving the circulating MOPCs in the osteogenic processes, such as signals that recruit the circulating MOPCs to the osseous tissues, have been obscure. In this report, we demonstrated for the first time that the MOPCs were mobilized from intact bones to transiently occupy approximately 80% of the mononuclear cell population in the circulating blood by BMP-2-pellet implantation. The mobilized MOPCs in the circulation did not express the hematopoietic marker CD45 on their surface, but they expressed CD44 and CXCR4, receptors of osteopontin and stromal cell-derived factor-1 (SDF-1), respectively. The MOPCs isolated from the mouse peripheral blood showed the ability to be osteoblasts in vitro and in vivo. Furthermore, the MOPCs in the circulation efficiently migrated to the region of bone formation by chemoattraction of SDF-1 expressed in vascular endothelial cells and the de novo osteoblasts of the region. These data may provide a novel insight into the mechanism of bone formation involving MOPCs in circulating blood, as well as perspective on the use of circulating MOPCs to accelerate bone regeneration in the future.

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

	INTRODUCTION

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

 
Bone marrow contains hematopoietic stem cells and mesenchymal stem/progenitor cells (MPCs) that can differentiate into various mesenchymal tissues, such as bone, cartilage, fat, and muscle [1–3]. These cells are also found in various mesenchymal tissues [4], although the relationship between the marrow mesenchymal stem/progenitor cells (MMPCs) and extramarrow MPCs has not been fully understood. Marrow MPCs have been shown to engraft not only in bone marrow but also in multiple mesenchymal tissues after systemic infusion [3, 5], suggesting that circulating blood might be a natural route for MMPC migration to the mesenchymal tissues in vivo.

Previous studies have shown the existence of osteoblast-lineage cells in the circulating blood of various mammals, including humans [6–8]. The circulating osteoblast-lineage cells were shown to form bone in culture and in transplanted animals [6]. Studies have also reported more circulating osteoblast-lineage cells during the adolescent growth spurt than in adulthood [6]. However, the origin and the functional role of those osteoblastic cells in human circulation are unclear.

Bone morphogenetic protein (BMP)-2 and other members of the BMP family are well-known inducers of bone formation in vitro and in vivo [9]. In the process of a fracture healing, BMP stimulation recruits MPCs to the fracture lesion and induces their differentiation into osteoblasts. An experimental model has also indicated that BMP-2 stimulation is essential for ectopic bone formation when BMP-2 is transplanted in the back muscles of mice [10]. Recently, we reported that marrow-derived osteoblast progenitor cells (MOPCs) in circulating blood participated in BMP-2-induced ectopic bone formation [11]. If circulating MOPCs play a major role in bone regeneration in vivo, efficient recruitment of MOPCs from bone marrow to the lesion seems to be critical to obtain mature and sufficient regeneration. However, the character of circulating MOPCs and precise mechanisms involving the MOPCs in the osteogenic processes, such as signals that recruit circulating MOPCs to the osseous tissues, have been obscure.

In this study, we characterized MOPCs in the circulating blood without expansion in culture and showed that MOPCs were mobilized in the circulation after stimulation with tissue injury, migrated to damaged tissues by chemoattraction of stromal cell-derived factor-1 (SDF-1), and provided a significant number of mature osteoblasts with BMP-2 stimulation during bone formation. We believe these findings provide novel insights into bone regeneration involving circulating MOPCs.

	MATERIALS AND METHODS

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

 
Bone Marrow Transplantation
Under sterile conditions, bone marrow cells were isolated from 8- to 10-week-old male C57BL/6 transgenic mice that ubiquitously expressed enhanced green fluorescent protein (GFP) [12]. Eight- to 10-week-old female C57BL/6 mice were lethally irradiated with 10 Gy. For total bone marrow transplantation (BMT), each irradiated recipient received 5 x 10⁶ bone marrow cells from GFP transgenic mice. For CD45/GFP double-positive BMT, the CD45-positive bone marrow cells of GFP transgenic mice were sorted using the magnetic cell sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Each irradiated recipient received 4.5 x 10⁶ CD45-positive marrow cells of GFP transgenic mice in combination with 0.5 x 10⁶ CD45-negative marrow cells of wild-type mice. All BMT mice were used at least 6 weeks after BMT. All animals were handled according to approved protocols and the guidelines of the Animal Committee of Osaka University.

Parabiotic Mouse Model
The parabiotic mouse model was generated as previously described [13]. A total BMT mouse and a wild-type mouse (C57/BL6) were sutured from the olecranon to the knee joint on the corresponding lateral aspects.

Preparation and Implantation of BMP-2-Containing Collagen Pellets
Recombinant human BMP-2 was provided by Astellas Pharma Inc. (Tokyo, http://www.astellas.com). The BMP-2 was suspended in buffer solution (5 mmol/l glutamic acid, 2.5% glycine, 0.5% sucrose, and 0.01% Tween 80, pH 4.5) at a concentration of 1 µg/µl. Next, 3 µl (3 µg of BMP-2) of the BMP-2 solution was diluted in 22 µl of phosphate-buffered saline (PBS) and blotted into a porous collagen disc (6 mm diameter, 1 mm thickness), freeze-dried, and stored at –20°C. All procedures were carried out under sterile conditions. BMP-2-containing or control PBS-containing collagen pellets were implanted on the backs of BMT mice, parabiotic mice, C57BL/6 mice, or nude mice. Three weeks later, fluorescent photos of ectopic bones were taken using a digital microscope (Multiviewer system VB-S20; Keyence, Osaka, Japan, http://www.keyence.com).

Immunohistochemistry and Analysis
The ectopic bones were removed and fixed with 4% paraformaldehyde at 4°C for 48 hours. After soft x-ray photos were taken, bones were decalcified with EDTA solution at 4°C for 6 days. The EDTA solution was changed every other day. After decalcification, the pellets were equilibrated in PBS containing 15% sucrose for 12 hours and then in PBS containing 30% sucrose for 12 hours, embedded in Tissue-Tek OCT Compound (Sakura Finetek, Tokyo, http://www.sakuraeu.com), frozen on dry ice, and stored at –20°C.

For immunofluorescence staining, 6-µm-thick sections were cut with a cryostat (Leica Microsystems AG, Wetzlar, Germany, http://www.leica.com). After washing, the sections were treated with 0.1% trypsin (Difco Laboratories, Detroit, MI, http://www.bd.com/ds) in PBS for 30 minutes at 37°C to activate antigens. Then, those sections were blocked with normal goat serum for 1 hour before incubation with polyclonal anti-mouse osteocalcin antibody (1:250; Takara Bio, Shiga, Japan, http://www.takara-bio.com) or polyclonal anti-mouse SDF-1a antibody (1:250; eBioscience Inc., San Diego, http://www.ebioscience.com). Subsequently, sections were stained with Alexa Fluor 546 goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) for 2 hours. Then, sections were stained with 4',6-diamidino-2-phenylindole (DAPI) for 10 minutes at room temperature and mounted with the antifade solution Vectashield (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

For staining endothelial progenitor cells and tissues around the pellets, the pellets were removed daily until day 7 after implantation. After removal, pellets were embedded in Tissue-Tek OCT Compound and frozen on dry ice. Six-micrometer-thick sections were blocked with normal goat serum for 1 hour before incubation with monoclonal anti-mouse CD31 antibody (1:250; BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/index_us.shtml), monoclonal anti-mouse CD34 antibody (1:100; BD Pharmingen), monoclonal anti-smooth-muscle actin antibody (1:250; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), or polyclonal anti-mouse SDF-1a antibody (1:250; eBioscience). Subsequently, sections were stained with Alexa Fluor 546 goat anti-rat IgG secondary antibody, Alexa Fluor 488 anti-rat IgG secondary antibody, or Alexa Fluor 488 anti-mouse IgG secondary antibody (Molecular Probes) with M.O.M. Kit (Vector Laboratories) for 2 hours. The sections were mounted with antifade solution Vectashield after 10 minutes of DAPI staining. All pictures were taken with a confocal laser microscope, model Radiance 2100 using LaserSharp 2000 software (Bio-Rad Japan, Tokyo, http://www.bio-rad.com). To assess the frequency of MOPCs for osteoblast differentiation, we counted the number of GFP-positive cells in the osteocalcin-positive osteoblasts lining the trabecular bone. The ratio was quantitatively calculated in at least five low-power visual fields.

Peripheral Blood Mononuclear Cell Isolation
Peripheral blood was taken from the heart with a 24-gauge needle and 1-ml syringe containing heparin and enriched for low-density mononuclear cells by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden, http://www.amersham.com) centrifugation. Red blood cells were removed by resuspending in 0.125% Tris-NH₄Cl buffer and sieving through a nylon mesh. Isolated peripheral blood mononuclear cells (PBMNCs) from BMP-2 pellet-implanted mice were reacted with anti-mouse CD45 microbeads (Miltenyi Biotec), and the CD45-negative cells dominantly containing MOPCs were sorted using the MidiMACS system (Miltenyi Biotec) according to the manufacturer's protocol.

In Vitro Differentiation
For induction of osteoblast differentiation in culture, MACS-sorted CD45-negative PBMNCs from BMP-2-implanted GFP transgenic mice were plated on a 24-well plate. The sorted cells were then inoculated in basal medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml streptomycin/penicillin, and 50% conditioned culture medium (DMEM with 10% FCS) of mouse bone marrow mesenchymal cells as a growth factor supplement (S. Otsuru and K. Tamai, unpublished data). To induce osteoblast differentiation, 300 ng/ml BMP-2 was added to the culture medium for 3 weeks. In some experiments, the sorted CD45-negative PBMNCs were cultured in the osteogenic medium consisting of Iscove's modified Dulbecco's medium supplemented with 0.1 µM dexamethasone (Nacalai Tesque Inc., Kyoto, Japan, http://www.nacalai.co.jp/en), 10 mM β-glycerol phosphate (Sigma-Aldrich), and 0.05 mM ascorbic acid 2-phosphate (Sigma-Aldrich) for 3–4 weeks.

Alizarin Red S Staining
To observe calcium deposition, cells were fixed with 4% paraformaldehyde and stained with 2% alizarin red S (Nacalai Tesque) solution in water for 10 minutes. Excess stain was removed by several washes with distilled water.

Alkaline Phosphatase Assay
Alkaline phosphatase (ALP) activity was assessed as previously described [14]. Cell lysates were centrifuged, and supernatants were used for the enzyme assay. Alkaline phosphatase activity was measured according to the methods of Kind-King, using a test kit (Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english) with phenylphosphate as a substrate. Enzyme activity was expressed in King-Armstrong units, normalized to protein concentration.

In Vivo Bone Forming Assay
Fully open interconnected porous calcium hydroxyapatite ceramics were synthesized by adopting a "foam-gel" technique from a slurry of hydroxyapatite (60% wt/wt) with a cross-linking substrate (polyethyleneimine, 40% wt/wt) as previously reported [15]. Blocks of the ceramics were cut and shaped into 5-mm-diameter disks that were 2 mm thick. Cell culturing in the pores of the ceramics was performed as previously reported [16, 17]. The ceramic disk was soaked in 200 µl of CD45-negative cultured PBMNC suspension from GFP-transgenic mice in normal medium (10⁶ cells per milliliter). After overnight incubation in a 96-well plate, normal medium was changed to osteogenic medium. The medium was renewed three times a week, and the cultures were maintained for 2 weeks. After a wash with PBS, the disks were implanted under the muscular fascia in the backs of nude mice. Disks without cells were also implanted as controls. Eight weeks later, the disks were harvested and fixed in 4% paraformaldehyde. After decalcification with K-CX (Falma Co., Osaka, Japan; http://www.falma.co.jp), the disks were embedded in paraffin, and the sections were stained with hematoxylin and eosin.

For immunofluorescence staining, the sections were treated with 0.1% trypsin (Difco Laboratories) in PBS for 30 minutes at 37°C to activate antigens. Next, those sections were blocked with normal goat serum for 1 hour before incubation with polyclonal anti-GFP antibody (1:250; MBL International Corp., Nagoya, Japan, http://www.mblintl.com). Subsequently, sections were stained with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Molecular Probes) for 2 hours. Sections were then stained with DAPI for 10 minutes at room temperature and mounted with the antifade solution Vectashield (Vector Laboratories).

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
Total RNA was prepared with an RNeasy Kit (Qiagen, Tokyo, http://www1.qiagen.com) according to the manufacturer's protocol. Reverse transcription was performed by conventional protocols with Superscript reverse transcriptase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and polymerase chain reaction (PCR) amplification was performed using the following primer sets: Cbfa1 (NM_009820 [GenBank] , 289 base pairs [bp]), 5'-CCGCACGACAACCGCACCAT-3' (forward) and 5'-CGCTCCGGCCCACAAATCTC-3' (reverse); osteopontin (NM_009263, 437 bp), 5'-TCACCATTCGGATGAGTCTG-3' (forward) and 5'-ACTTGTGGCTCTGATGTTCC-3' (reverse); ALP (NM_007431, 180 bp), 5'-CGCCAGAGTACGCTCCCGCC-3' (forward) and 5'-TGTACCCTGAGATTCGT-3' (reverse); osteocalcin (X04142, 350 bp),5'-CTGACCTCACAGATCCCAAG-3' (forward) and 5'-GGAGCTGCTGTGACATCC-3' (reverse); and SDF-1 (NM_021704, 538 bp), 5'-ACGCCAAGGTGGTCGCCGTGCTGG-3' (forward) and 5'-GTTAGGGTAATACAATTCCTTAGA-3' (reverse).

Flow Cytometry
Isolated PBMNCs were suspended in 100 µl of PBS containing fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD45; phycoerythrin (PE)-conjugated anti-mouse CD11b, CD31, CD34, CD44, Flk-1, and Sca-1 (BD Pharmingen); and biotin-conjugated anti-mouse Gr-1 and CXCR4 (BD Pharmingen). Cells were then incubated for 30 minutes at 4°C in the dark. Subsequently, cells were stained with Streptavidin PE or Streptavidin PerCP (BD Pharmingen) or anti-rat IgG secondary antibody or anti-goat IgG secondary antibody (Molecular Probes) for 30 minutes at 4°C in the dark.

For the time-course analysis of the CD45-negative population in PBMNCs, BMP-2 pellets were implanted on the backs of 8- to 10-week-old female C57BL/6 mice daily, one mouse per day, for 7 days. At day 7, blood samples were taken, and hemolyzed PBMNCs were harvested. The harvested PBMNCs were reacted with FITC-conjugated anti-mouse CD45. Flow cytometry analysis was performed with a FACScan instrument using CellQuest software (Becton, Dickinson and Company, San Diego, http://www.bd.com).

In Vitro Migration Assay
After different concentrations of SDF-1a (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) were added to the lower chamber, the 5 x 10⁵ isolated CD45-negative PBMNCs from BMP-2 pellet-implanted mice in 100 µl of DMEM without growth factors were applied to the upper chamber of the membrane of the 96-well cell migration kit (Chemicon, Temecula, CA, http://www.chemicon.com). Some cells were pretreated with a CXCR4-blocking antibody (2B11; BD Pharmingen). After 4 hours of incubation at 37°C, the migratory cells on the bottom of the insert membrane were dissociated from the membrane by incubation with cell detachment buffer. These cells were stained with CyQuant GR dye (Molecular Probes, Eugene, OR, http://probes.invitrogen.com), and the fluorescence was measured with a fluorescence plate reader.

In Vivo Migration Assay
For the transplantation experiment, nude mice implanted with BMP-2-containing collagen pellets were injected via a tail vein with sorted CD45-negative PBMNCs with or without CXCR4-blocking antibody (2B11; BD Pharmingen) pretreatment from the GFP-transgenic BMP-2-implanted mice for 7 days.

Real-Time PCR
Primers and probes for hypoxia inducible factor-1 (HIF-1), SDF-1, and glyceraldehyde-3-phosphate dehydrogenase were purchased from Applied Biosystems (Foster City, CA, http://www.appliedbiosystems.com). Real-time PCR was carried out and measured by the ABI Prism 7900HT Sequence Detection System using SDS 2.2 software (Applied Biosystems).

Statistical Analysis
All experiments were repeated four to seven times. Statistical analyses were performed with the unpaired t test or the paired Student t test. p values <0.05 were considered statistically significant.

	RESULTS

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

 
Bone Marrow-Derived Osteoblast Progenitor Cells in Circulation Contribute to BMP-2-Induced Ectopic Bone Formation
We have already reported that osteoblast progenitor cells (OPCs) were recruited from bone marrow to the circulating blood and contributed to the BMP-2-induced ectopic bone formation [11]. To obtain more direct evidence that circulating MOPCs were recruited to the region of the BMP-2 pellet to generate ectopic bone, we established a mouse model with parabiotic pairings that shared a circulatory system between a wild-type mouse and a GFP-BMT mouse (Fig. 1A) [13]. In our parabiotic mouse model, a wild-type mouse was surgically connected with a GFP-BMT mouse whose bone marrow had been replaced by GFP-transgenic bone marrow cells. The wild-type mouse can receive bone marrow-derived GFP-positive circulating cells from the GFP-BMT mouse after they develop shared circulation in a few weeks. We implanted a BMP-2 pellet into the wild-type mouse of the parabiotic pairings (Fig. 1A). Three weeks after transplantation, GFP fluorescence was detected at the region where the ectopic bone had formed (Fig. 1B). Histologic analysis revealed that 24.5% ± 3.1% of the osteoblasts that aligned on the regenerating bone and expressed osteocalcin (OC) were GFP-positive cells that originated from the bone marrow of the GFP-BMT mouse (Fig. 1C; supplemental online Fig. 1). If both mice provide the bone marrow cells equally, these data suggest that approximately 50% of the regenerating osteoblasts may be derived from endogenous circulating MOPCs in parabiotic mice.

View larger version (66K): [in this window] [in a new window]   Figure 1. Bone marrow-derived osteoblast progenitor cells contribute to BMP-2-induced ectopic bone formation via circulation in a parabiotic mouse. (A): Parabiotic pairing between a GFP-BMT mouse and a wild-type mouse. A BMP-2 pellet was implanted into the wild-type mouse that could receive GFP-positive bone marrow cells from the GFP-BMT mouse through the circulation. (B): A BMP-2 pellet showed accumulation of GFP fluorescence 3 weeks after implantation under the muscular fascia of a wild-type parabiotic mouse. A soft x-ray photo of the BMP-2 pellet 3 weeks after the implantation demonstrated that ectopic bone formed in the BMP-2 pellet. Histologic section stained with H&E of the BMP-2 pellet 3 weeks after implantation also revealed bone formation in the BMP-2 pellet. Magnification, x200. (C): Immunofluorescence staining of the boxed region in the H&E section showed that the cells lining the newly generated bone were osteoblasts expressing OC. Some of those osteoblasts expressing osteocalcin also exhibited GFP fluorescence (arrowheads). Magnification, x600. Abbreviations: BMP, bone morphogenetic protein; BMT, bone marrow transplantation; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; OC, osteocalcin.

View larger version (47K): [in this window] [in a new window]   Figure 2. CD45-negative fraction of bone marrow cells predominantly participated in the BMP-2-induced ectopic bone formation. (A): CD45-negative bone marrow cells from wild-type mice and CD45-positive bone marrow cells from GFP-transgenic mice were transplanted into a lethal-dose-irradiated wild-type mouse (CD45/GFP-BMT) before the BMP-2-pellet implantation. (B): The ectopic bone (circled with a dotted line) in the CD45/GFP-BMT mouse showed weak GFP fluorescence. A soft x-ray photo and H&E-stained histologic section showed successful ectopic bone formation in the CD45/GFP-BMT mice. Immunofluorescence staining revealed fewer GFP-positive cells in the ectopic bone of the CD45/GFP-BMT mice than in the ectopic bone of the GFP-BMT mice. Magnification, x400. (C): Total bone marrow cells from GFP transgenic mice were transplanted to lethally irradiated wild-type mice (GFP-BMT mouse). A BMP-2 pellet in the GFP-BMT mouse showed stronger GFP fluorescence (circled with a dotted line). A soft x-ray photo and histologic H&E-stained section showed bone formation in the BMP-2 pellet in the GFP-BMT mice as well. Immunofluorescence staining revealed that more GFP-positive cells expressed OC in the newly formed bone. Magnification, x200. (D): Quantitative analysis showed that the percentage of GFP-positive/osteocalcin-positive osteoblasts in the osteocalcin-positive osteoblasts lining the trabecular bone significantly decreased in the CD45/GFP-BMT mice compared with the GFP-BMT mice. *, p = .00127. Abbreviations: BMC, bone marrow cell; BMP, bone morphogenetic protein; BMT, bone marrow transplantation; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; OC, osteocalcin.

View larger version (53K): [in this window] [in a new window]   Figure 3. MOPCs were detected in PBMNCs from BMP-2 pellet-implanted mice. (A): Representative results of time-course analysis of five different sets of experiments. The CD45-negative cell population (%) in PBMNCs and BMCs of the BMP-2-implanted mice was analyzed with flow cytometry for 7 days after BMP-2 implantation. Robust but transient appearance of a CD45-negative population in PBMNCs according to the significant reduction of the CD45-negative population in BMCs within 7 days after BMP-2-implantation was observed in five different sets of experiments compared with nontreated wild-type mice (day 0). (B): Analysis of average percentage of CD45-negative population in PBMNCs at peak time within 7 days after BMP-2 implantation in five different sets of experiments showed that the CD45-negative population in PBMNCs was significantly induced by BMP-2-pellet implantation. *, p = .0000142. (C): Reverse transcription-polymerase chain reaction analysis of the magnetic cell sorting-sorted CD45-negative PBMNCs showed that these cells exhibited Cbfa1 expression before culture (lane 1), additional OP expression in culture without BMP-2 stimulation (lane 2), and ALP and OC expression in culture with BMP-2 stimulation for 3 weeks (300 ng/ml; lane 3). (D): The sorted CD45-negative cells cultured in basal medium and in osteogenic medium for 4 weeks showed morphogenic changes to osteoblastic features. Magnification, x40. Alizarin red staining (right panels) showed that calcium deposition was observed only in cells cultured in osteogenic medium. Magnification, x40. (E): ALP assay showed that ALP activity was increased when the CD45-negative cells in PBMNCs were cultured in osteogenic medium. (F): Histologic H&E-stained sections of the hydroxyapatite transplanted in vivo with (right) or without (left) the CD45-negative cells in PBMNCs revealed that those cells could form bone in hydroxyapatite. Magnification, x100. Abbreviations: ALP, alkaline phosphatase; BMC, bone marrow cell; BMP, bone morphogenetic protein; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; K-A, King-Armstrong; OC, osteocalcin; OP, osteopontin; PBMNC, peripheral blood mononuclear cell.

Characterization of Circulating MOPCs
We further analyzed cell surface markers of the circulating MOPCs in PBMNCs by flow cytometry analysis (Fig. 4). Significant expression of CD44, which is expressed in mesenchymal cells as a receptor of OP [18], was observed (Fig. 4). However, neither hematopoietic lineage markers, such as CD45, CD11b, or Gr-1, nor endothelial lineage markers, such as CD34, Flk-1, or CD31, were detected. Interestingly, circulating CD45-negative MOPCs markedly expressed CXCR4 (Fig. 4), a receptor of the chemokine SDF-1 [19]. The SDF-1 chemokine is known to hold CXCR4-positive stem cells in the bone marrow niche [20–22] and to recruit those cells to peripheral tissues that express SDF-1 [23, 24].

View larger version (33K): [in this window] [in a new window]   Figure 4. Flow cytometry analysis of marrow-derived osteoblast progenitor cells (MOPCs). Most of the peripheral blood mononuclear cells (PBMNCs) in wild-type mice (control) were CD45-positive. CD45-negative MOPCs were increased in PBMNCs of BMP-2 pellet-implanted mice on day 4. Endothelial lineage markers (CD34, CD31, and Flk1) and hematopoietic lineage markers (CD45, CD11b, and Gr-1) were not detected in the CD45-negative MOPCs in BMP-2-implanted mice. CD44 and CXCR4 were highly expressed in the CD45-negative MOPCs of BMP-2-implanted mice compared with the PBMNCs from wild-type mice. Abbreviation: BMP, bone morphogenetic protein.

View larger version (69K): [in this window] [in a new window]   Figure 5. SDF-1 was expressed around the BMP-2 P. (A): Histologic H&E-staining and immunofluorescence staining showed that vessels around the BMP-2 P highly expressed SDF-1. Magnification, x100 (first) and x500 (second). Immunofluorescence staining of the serial sections of the SDF-1-positive section around the BMP-2 P showed that SDF-1 was expressed from the CD31-, CD34-, and SMA-positive vessel. Magnification, x500. (B): The quantitative time-course analysis of SDF-1 and hypoxia inducible factor-1 (HIF-1) mRNA expression in the BMP-2 and collagen Ps revealed that SDF-1 and HIF-1 expression increased significantly in BMP-2 Ps compared with control tissue (day 0). *, p < .01. The high expression was significantly maintained on day 7 in BMP-2 Ps compared with those on day 7 in collagen Ps. *, p < .01. The fold increases of expression levels were normalized to those of control tissue (day 0). (C): The immunofluorescence staining of the BMP-2-induced ectopic bone on day 14 demonstrated osteoblasts lining the newly formed bone expressed SDF-1. Magnification, x500. Abbreviations: BMP, bone morphogenetic protein; DAPI, 4',6-diamidino-2-phenylindole; P, pellet; SDF, stromal cell-derived factor; SMA, smooth muscle actin.

View larger version (40K): [in this window] [in a new window]   Figure 6. MOPCs in the circulation migrate to the bone formation site in the CXCR4/SDF-1 pathway. (A): Effect of SDF-1 on MOPCs in the circulation was checked in the Boyden chamber migration assay. The MOPC migration was stimulated by SDF-1. *, p < .05. The effect of SDF-1 was significantly decreased when the MOPCs were pretreated with CXCR4-blocking antibody. **, p < .05. The number of migrated cells was measured as RFU. (B): MOPCs from a BMP-2-implanted GFP transgenic mice were pretreated with or without the CXCR4-blocking antibody and were injected to a BMP-2-implanted nude mouse daily for 7 days. Immunofluorescence staining of ectopic bone from the nude mouse showed that injected GFP-positive MOPCs differentiated to OC-positive osteoblasts lining the newly formed trabecular bone. Fewer GFP- and OC-positive osteoblasts were found in sections of ectopic bone from a nude mouse injected daily for 7 days with the GFP transgenic MOPCs pretreated with the CXCR4-blocking antibody. Magnification, x400. (C): Quantitative analysis of GFP-positive/OC-positive osteoblasts lining the trabecular bone showed that the MOPC migration was significantly blocked by blocking CXCR4. *, p = .00019. Abbreviations: RFU, relative fluorescence units; ab, antibody; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; i.v., intravenous; MOPC, marrow-derived osteoblast progenitor cell; OC, osteocalcin; SDF, stromal cell-derived factor.

	DISCUSSION

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

Recently, we reported that marrow cells in intact bone are a major, if not exclusive, source of circulating OPCs under a BMP-2-induced ectopic bone-forming condition in mice, and these cells endogenously participate in the process of the ectopic bone formation [11]. In this study, a parabiotic pairing mouse model showed that 50% of all osteoblasts were derived from MOPCs in the BMP-2-induced ectopic bone. Previous studies of fracture healing have shown that fracture stimulation induces BMP-2 expression in the surrounding tissues [27–29], suggesting that endogenously circulating MOPCs may also contribute to fracture healing, as well as to ectopic bone formation.

Circulating MOPCs seem to present as a small population without induction. Even after inducing stimulation, the increase of circulating MOPCs was time-limited and not maintained for more than few days. The peak of the MOPC induction in circulation oscillated between day 3 and day 7 after BMP-2 implantation, possibly because of the strength of signals generated by both BMP-2 and injury stimulation (data not shown). These findings may explain the previous difficulties in detecting circulating mesenchymal cells without expansion in culture [30, 31].

SDF-1 has been characterized as a potent CXC chemokine that is constitutively expressed in various cell types, including mesenchymal stem cells and osteoblasts, in bone marrow, and in dermal and synovial fibroblasts [22, 32, 33]. SDF-1 retains hematopoietic stem cells that express CXCR4, a receptor for SDF-1, in bone marrow [21, 22]. SDF-1 expressed in peripheral tissues under inflammatory conditions recruits circulating lymphocytes, monocytes, and other hematopoietic cells, except neutrophils, to the peripheral tissues via the CXCR4/SDF-1 system [34, 35]. A recent study showed that SDF-1 in mural cells around blood vessels functioned to entrap bone marrow-derived vascular endothelial progenitor cells, which express CXCR4, in circulation [36]. We demonstrated significant expression of CXCR4 on circulating MOPCs. A strong expression of SDF-1 was noted not in the circulating MOPCs (supplemental online Fig. 4) but in vascular endothelial cells and osteoblasts in the regions of the ectopic osteogenesis, suggesting that the CXCR4/SDF-1 system may play an important role in entrapping circulating MOPCs around the area of the bone formation, although factors besides SDF-1 may also contribute to the osteogenic processes with MOPCs recruitment. Further analysis showed that elevations of SDF-1 levels were accompanied by upregulation of HIF-1, a well-known transcriptional factor that upregulates SDF-1 expression. HIF-1 and SDF-1 induction were obtained by implantation of the collagen pellet without BMP-2 and probably resulted from the hypoxic tissue damage induced by surgical implantation of the pellet. The BMP-2 pellet, however, exhibited significantly prolonged expression of HIF-1 and SDF-1 at day 7 compared with the collagen pellet itself. This sustained expression of SDF-1 in the BMP-2-pellet was further confirmed in osteoblasts, as well as in vascular endothelial cells of the newly generating bone after day 7 (data not shown). Collectively, HIF-1-dependent initial expression of SDF-1 in arterioles around the BMP-2 pellet seemed to entrap circulating MOPCs, followed by BMP-2-dependent recruitment and differentiation of the trapped MOPCs to osteoblasts, which expressed SDF-1 and may have further enhanced bone formation by continuous recruitment of MOPCs to the osseous tissue in collaboration with the CXCR4/SDF-1 pathway. Previous studies [37, 38] also showed that the CXCR4/SDF-1 pathway plays a pivotal role in the migration of stem cells to regenerating tissues, suggesting that the hypoxic condition induced by tissue injury plays a role in the induction of SDF-1 expression at the initial stage of tissue regeneration.

Recent studies have indicated that CD44 binds to the ubiquitous matrix protein OP and serves as a receptor on CD44-expressing cells to bind to OP [18]. Osteopontin is known to be expressed in osteoblasts and secreted in the areas of the callus formation [39], suggesting that OP functions as the major ligand for CD44 on migrating osteoblast progenitor cells in the remodeling phase of fracture healing [40]. In this context, it is interesting to note that circulating MOPCs significantly expressed CD44 on the cell surface. Interaction between OP and CD44 on MOPCs may be important for the acceleration of bone formation in combination with the CXCR4/SDF-1 system and BMP stimulation.

Signals that trigger migration of the particular cell population from bone marrow to the circulation were not identified in this study. Vascular endothelial growth factor (VEGF) was previously shown to be sufficient for recruitment of marrow-derived vascular endothelial progenitor cells into the circulation [36]. We also observed elevation of VEGF levels in muscular tissue around the implanted collagen pellet (data not shown). This observation may suggest that VEGF contributes to angiogenesis in the area of bone regeneration, although further evidence must be obtained to support this conclusion. Implantation of the collagen pellet itself induced a significant number of MOPCs in the circulation, suggesting that surgical injury may induce production of MOPC-recruiting signals, probably because of hypoxic stress in injured tissue, as previously reported [41]. BMP-2-pellet implantation, however, induced a relatively higher increase in MOPCs in the circulation compared with empty collagen pellets. In addition, subcutaneous injection of BMP-2 without extensive tissue damage could induce 20% of CD45-negative cells in circulation (data not shown), suggesting that both BMP-2 and tissue injury contribute to the mobilization of MOPCs in circulation. A lack of detectable expression of bone morphogenetic protein receptor II (BMPR-II) on the MOPCs in the circulation (supplemental online Fig. 5) suggests that BMP-2 does not participate in MOPC mobilization but that other factors induced by BMP-2 may. This hypothesis may be supported by our observation that there were no significant changes in the concentration of BMP-2 in serum between wild-type nontreated mice and BMP-2 pellet-implanted mice (supplemental online Fig. 5). Furthermore, we could establish a C57BL/6 mouse bone marrow-derived stromal cell line, which had been shown to be negative for BMPR-II but maintained the capability to differentiate to mature mineralizing osteoblasts when cultured in osteogenic medium (S. Otsuru et al., unpublished data), suggesting that BMPR-II expression may not be essential to maintain osteogenic features in the initial undifferentiated condition of the MOPCs.

The importance of providing additional OPCs to the site of bone formation has been shown by a number of previous studies [42–47]. Identification of signals that induce migration of MOPCs in the circulation may have clinical applications in the future, as the ability to increase MOPCs in the circulation may help patients with intractable bone fractures by inducing further accumulation of MOPCs to the fractured lesion. Robust induction of circulating MOPCs may also enable us to easily isolate these cells by simple blood sampling, providing the possibility to develop novel cell-based regenerative therapies for intractable bone fractures and possibly for other damaged tissues. Because current procedures to isolate cells directly from bone marrow are invasive, the easy isolation of MOPCs from peripheral blood has advantages in terms of safety, repeatability, and acceptability. Genetic manipulation of isolated MOPCs may also have possible applications in the treatment of genetic disorders such as osteogenesis imperfecta [48, 49].

The potency of circulating MOPCs as stem cells is another issue to be addressed in future studies. Because Sca-1 is an established marker of both mesenchymal and hematopoietic stem cells, MOPCs with low levels of Sca-1 expression seem to have different features compared with stem cells in bone marrow [50, 51]. Demonstration of efficient differentiation activities of MOPCs, in addition to osteoblastic lineage, may illustrate the additional importance of these cells in tissue regeneration.

	CONCLUSION

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

 
We report here the crucial role of the CXCR4/SDF-1 pathway in the bone formation involving circulating MOPCs. The mobilized MOPCs that expressed CXCR4 were recruited to the bone-forming site by SDF-1 expressed in vascular endothelial cells and the de novo osteoblasts of the region. These data may provide perspective on the use of circulating MOPCs to accelerate bone regeneration in the future.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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

 
This work was supported by the Northern Osaka (Saito) Biomedical Knowledge-Based Cluster Creation Project and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	REFERENCES

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1. Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.[CrossRef][Medline]

2. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.[Abstract/Free Full Text]

3. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–74.[Abstract/Free Full Text]

4. Minguell JJ, Erices A, Conget P. Mesenchymal stem cells. Exp Biol Med (Maywood) 2001;226:507–520.[Abstract/Free Full Text]

5. Pereira RF, Halford KW, O'Hara MD et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci U S A 1995;92:4857–4861.[Abstract/Free Full Text]

6. Eghbali-Fatourechi GZ, Lamsam J, Fraser D et al. Circulating osteoblast-lineage cells in humans. N Engl J Med 2005;352:1959–1966.[Abstract/Free Full Text]

7. Kuznetsov SA, Mankani MH, Gronthos S et al. Circulating skeletal stem cells. J Cell Biol 2001;153:1133–1140.[Abstract/Free Full Text]

8. Wan C, He Q, Li G. Allogenic peripheral blood derived mesenchymal stem cells (MSCs) enhance bone regeneration in rabbit ulna critical-sized bone defect model. J Orthop Res 2006;24:610–618.[CrossRef][Medline]

9. Wozney JM, Rosen V, Celeste AJ et al. Novel regulators of bone formation: Molecular clones and activities. Science 1988;242:1528–1534.[Abstract/Free Full Text]

10. Takaoka K, Nakahara H, Yoshikawa H et al. Ectopic bone induction on and in porous hydroxyapatite combined with collagen and bone morphogenetic protein. Clin Orthop Relat Res 1988;250–254.

11. Otsuru S, Tamai K, Yamazaki T et al. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice. Biochem Biophys Res Commun 2007;354:453–458.[CrossRef][Medline]

12. Okabe M, Ikawa M, Kominami K et al. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319.[CrossRef][Medline]

13. Wright DE, Wagers AJ, Gulati AP et al. Physiological migration of hematopoietic stem and progenitor cells. Science 2001;294:1933–1936.[Abstract/Free Full Text]

14. Wakabayashi S, Tsutsumimoto T, Kawasaki S et al. Involvement of phosphodiesterase isozymes in osteoblastic differentiation. J Bone Miner Res 2002;17:249–256.[CrossRef][Medline]

15. Tamai N, Myoui A, Tomita T et al. Novel hydroxyapatite ceramics with an interconnective porous structure exhibit superior osteoconduction in vivo. J Biomed Mater Res 2002;59:110–117.[CrossRef][Medline]

16. Nishikawa M, Myoui A, Ohgushi H et al. Bone tissue engineering using novel interconnected porous hydroxyapatite ceramics combined with marrow mesenchymal cells: Quantitative and three-dimensional image analysis. Cell Transplant 2004;13:367–376.[Medline]

17. Nishikawa M, Ohgushi H, Tamai N et al. The effect of simulated microgravity by three-dimensional clinostat on bone tissue engineering. Cell Transplant 2005;14:829–835.[Medline]

18. Weber GF, Ashkar S, Glimcher MJ et al. Receptor-ligand interaction between CD44 and osteopontin (Eta-1). Science 1996;271:509–512.[Abstract]

19. Bleul CC, Farzan M, Choe H et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 1996;382:829–833.[CrossRef][Medline]

20. Dar A, Goichberg P, Shinder V et al. Chemokine receptor CXCR4-dependent internalization and resecretion of functional chemokine SDF-1 by bone marrow endothelial and stromal cells. Nat Immunol 2005;6:1038–1046.[CrossRef][Medline]

21. Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 1999;10:463–471.[CrossRef][Medline]

22. Nagasawa T, Hirota S, Tachibana K et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996;382:635–638.[CrossRef][Medline]

23. Ceradini DJ, Kulkarni AR, Callaghan MJ et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 2004;10:858–864.[CrossRef][Medline]

24. Ratajczak MZ, Majka M, Kucia M et al. Expression of functional CXCR4 by muscle satellite cells and secretion of SDF-1 by muscle-derived fibroblasts is associated with the presence of both muscle progenitors in bone marrow and hematopoietic stem/progenitor cells in muscles. STEM CELLS 2003;21:363–371.[Abstract/Free Full Text]

25. Fernandez M, Simon V, Herrera G et al. Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplant 1997;20:265–271.[CrossRef][Medline]

26. Roufosse CA, Direkze NC, Otto WR et al. Circulating mesenchymal stem cells. Int J Biochem Cell Biol 2004;36:585–597.[CrossRef][Medline]

27. Bostrom MP. Expression of bone morphogenetic proteins in fracture healing. Clin Orthop Relat Res 1998;S116–S123.

28. Bostrom MP, Lane JM, Berberian WS et al. Immunolocalization and expression of bone morphogenetic proteins 2 and 4 in fracture healing. J Orthop Res 1995;13:357–367.[CrossRef][Medline]

29. Ishidou Y, Kitajima I, Obama H et al. Enhanced expression of type I receptors for bone morphogenetic proteins during bone formation. J Bone Miner Res 1995;10:1651–1659.[Medline]

30. Lazarus HM, Haynesworth SE, Gerson SL et al. Human bone marrow-derived mesenchymal (stromal) progenitor cells (MPCs) cannot be recovered from peripheral blood progenitor cell collections. J Hematother 1997;6:447–455.[Medline]

31. Wexler SA, Donaldson C, Denning-Kendall P et al. Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not. Br J Haematol 2003;121:368–374.[CrossRef][Medline]

32. Nagasawa T, Nakajima T, Tachibana K et al. Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin. Proc Natl Acad Sci U S A 1996;93:14726–14729.[Abstract/Free Full Text]

33. Zvaifler NJ, Marinova-Mutafchieva L, Adams G et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2000;2:477–488.[CrossRef][Medline]

34. Aiuti A, Webb IJ, Bleul C et al. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med 1997;185:111–120.[Abstract/Free Full Text]

35. Bleul CC, Fuhlbrigge RC, Casasnovas JM et al. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med 1996;184:1101–1109.[Abstract/Free Full Text]

36. Grunewald M, Avraham I, Dor Y et al. VEGF-induced adult neovascularization: Recruitment, retention, and role of accessory cells. Cell 2006;124:175–189.[CrossRef][Medline]

37. Imitola J, Raddassi K, Park KI et al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A 2004;101:18117–18122.[Abstract/Free Full Text]

38. Ji JF, He BP, Dheen ST et al. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury. STEM CELLS 2004;22:415–427.[Abstract/Free Full Text]

39. Hirakawa K, Hirota S, Ikeda T et al. Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization. J Bone Miner Res 1994;9:1551–1557.[Medline]

40. Yamazaki M, Nakajima F, Ogasawara A et al. Spatial and temporal distribution of CD44 and osteopontin in fracture callus. J Bone Joint Surg Br 1999;81:508–515.[CrossRef][Medline]

41. Rochefort GY, Delorme B, Lopez A et al. Multipotential mesenchymal stem cells are mobilized into peripheral blood by hypoxia. STEM CELLS 2006;24:2202–2208.[Abstract/Free Full Text]

42. Curylo LJ, Johnstone B, Petersilge CA et al. Augmentation of spinal arthrodesis with autologous bone marrow in a rabbit posterolateral spine fusion model. Spine 1999;24:434–438 discussion 438–439.[CrossRef][Medline]

43. Grundel RE, Chapman MW, Yee T et al. Autogeneic bone marrow and porous biphasic calcium phosphate ceramic for segmental bone defects in the canine ulna. Clin Orthop Relat Res 1991;244–258.

44. Lindholm TS, Nilsson OS, Lindholm TC. Extraskeletal and intraskeletal new bone formation induced by demineralized bone matrix combined with bone marrow cells. Clin Orthop Relat Res 1982;251–255.

45. Lindholm TS, Ragni P, Lindholm TC. Response of bone marrow stroma cells to demineralized cortical bone matrix in experimental spinal fusion in rabbits. Clin Orthop Relat Res 1988;296–302.

46. Takagi K, Urist MR. The role of bone marrow in bone morphogenetic protein-induced repair of femoral massive diaphyseal defects. Clin Orthop Relat Res 1982;224–231.

47. Werntz JR, Lane JM, Burstein AH et al. Qualitative and quantitative analysis of orthotopic bone regeneration by marrow. J Orthop Res 1996;14:85–93.[CrossRef][Medline]

48. Chamberlain JR, Schwarze U, Wang PR et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303:1198–1201.[Abstract/Free Full Text]

49. Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.[CrossRef][Medline]

50. Meirelles LS, Nardi NB. Murine marrow-derived mesenchymal stem cell: Isolation, in vitro expression, and characterization. Br J Haematol 2003;123:702–711.[CrossRef][Medline]

51. Baddoo M, Hill K, Wilkinson R et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J Cell Biochem 2003;89:1235–1249.[CrossRef][Medline]

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