Osteogenic Differentiation of Noncultured Immunoisolated Bone Marrow-Derived CD1 - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:04 |只看该作者 |倒序浏览 |打印

作者：Hadi Aslana, Yoram Zilbermana, Leonid Kandelb, Meir Liebergallb, Rod J. Oskouianc, Dan Gazita, Zulma Gazita作者单位：aSkeletal Biotechnology Laboratory, Hebrew UniversityCHadassah Medical Center, Jerusalem, Israel;bOrthopedic Surgery Department, The HadassahCHebrew University Medical School, Jerusalem, Israel;cNanoSpine, The Future of Spine Therapies, Charlottesville, Virginia, USA
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      【摘要】+ q1 N7 A/ ^; U, S3 X- B, e
   The culture expansion of human mesenchymal stem cells (hMSCs) may alter their characteristics and is a costly and time-consuming stage. This study demonstrates for the first time that immunoisolated noncultured CD105-positive (CD105 ) hMSCs are multipotent in vitro and exhibit the capacity to form bone in vivo. hMSCs are recognized as promising tools for bone regeneration. However, the culture stage is a limiting step in the clinical setting. To establish a simple, efficient, and fast method for applying these cells for bone formation, a distinct population of CD105  hMSCs was isolated from bone marrow (BM) by using positive selection based on the expression of CD105 (endoglin). The immunoisolated CD105  cell fraction represented 2.3% ¡À 0.45% of the mononuclear cells (MNCs). Flow cytometry analysis of freshly immunoisolated CD105  cells revealed a purity of 79.7% ¡À 3.2%. In vitro, the CD105  cell fraction displayed significantly more colony-forming units-fibroblasts (CFU-Fs; 6.3 ¡À 1.4) than unseparated MNCs (1.1 ¡À 0.3; p < .05). Culture-expanded CD105  cells expressed CD105, CD44, CD29, CD90, and CD106 but not CD14, CD34, CD45, or CD31 surface antigens, and these cells were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. In addition, freshly immunoisolated CD105  cells responded in vivo to recombinant bone morphogenetic protein-2 by differentiating into chondrocytes and osteoblasts. Genetic engineering of freshly immunoisolated CD105  cells was accomplished using either adenoviral or lentiviral vectors. Based on these findings, it is proposed that noncultured BM-derived CD105  hMSCs are osteogenic cells that can be genetically engineered to induce tissue generation in vivo. ( s; F# e( H5 ]4 K: @1 k7 ?
      【关键词】 Adult human mesenchymal stem cells Positive selection Genetic engineering Bone regeneration
   INTRODUCTION

In the development of stem cell-based therapeutic platforms for bone regeneration, the use of stem cells within the shortest period of time after their isolation will be substantially advantageous. We demonstrate in this report that human mesenchymal stem cells (hMSCs) can be immunoisolated and induced to differentiate into bone tissue without expansion.

Adult mesenchymal stem cells (MSCs) are considered one of the most promising tools for cell and cell-based gene therapy in bone repair .6 l9 W; Y7 c* c2 f

Several protocols were recently established to enable regeneration of large bone defects by using hMSCs that have been expanded in culture. These cells differentiate into osteogenic cells and, as vehicles, deliver a therapeutic gene product, such as bone morphogenetic proteins (BMPs) .

The culture expansion stage is extremely costly and time consuming, and in many cases the cells may lose their multipotentiality in vivo and fail to meet the desired goal. Rubio et al. reported that cultured hMSCs can undergo spontaneous transformation as a consequence of the in vitro expansion .

The isolation of an hMSC-enriched population requires an efficient and reproducible method. Few methods have been described for the isolation of MSCs, including enhancement of the plastic adherence property of the cells by using selected amounts of fetal calf serum (FCS) .
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In the present study, we have used the CD105-based immunoisolation method to obtain a fresh noncultured population of CD105  hMSCs and to determine their osteogenic potential both in vitro and in vivo. Our results demonstrate that this noncultured population of adult stem cells can be induced to undergo osteogenic differentiation in vivo and can be genetically modified; therefore, they could serve as an attractive therapeutic tool for bone regeneration purposes.) b/ E; j- j5 i) u0 U
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MATERIALS AND METHODS, g; y) q9 i( a; a& ], t& u3 a% |

Isolation of hMSCs from BM/ h% N7 }& @$ h( y# F

Immunomagnetic Isolation of CD105-Positive Cells. Human BM was recovered from heparinized trabecular bone samples collected from the proximal femora of patients undergoing corrective orthopedic surgery. Only donors (males and females) ages 18¨C80 years, excluding any malignancy, metabolic disorder, or infectious disease, were enrolled. (This study was approved by the Helsinki Committee Board of the Hadassah Medical Center, Jerusalem, Israel, and signed informed consent was obtained from each patient.) Each BM-containing trabecular bone sample was flushed with phosphate-buffered saline (1x PBS). To isolate mononuclear cells (MNCs), whole BM cells were layered over lymphocyte separation medium (ICN-Cappel Biomedicals, Inc., Aurora, OH) and centrifuged at 900g for 30 minutes at room temperature, without a break./ ]; L/ F- j& N" P- x% g
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Suspensions of MNCs obtained from BM were washed once with 1x PBS and twice with magnetic cell sorting (MACS) buffer (1x PBS containing 0.5% bovine serum albumin , pH 7.2). The cells were resuspended in MACS buffer at a concentration of 107 cells per 80 µl, transferred to a 1.5-ml test tube containing microbeads of directly conjugated mouse anti-human CD105 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), and placed on a rotator in the dark for 15 minutes at 4¡ãC. The cells were washed with 1x PBS, resuspended in MACS buffer, and separated using either a miniMACS or AutoMACS separation device and column (Miltenyi Biotec GmbH) according to the manufacturer¡¯s recommendation. To recover the CD105-positive (CD105 ) cells, the column was removed from the magnetic device, and the cells were flushed out with MACS buffer. The CD105-negative (CD105¨C) and CD105  cells were then recovered by centrifugation for future use. No differences in the yield, viability, or purity of the cells were observed when using the miniMACS and AutoMACS devices. However, the AutoMACS enabled separating higher numbers of MNCs in a faster manner.
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Cell Culture. Either MNCs or CD105¨C or CD105  cells were resuspended in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) that was supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 10% FCS (Biological Industries, Kibbutz Beit Haemek, Israel). The cells were then plated onto tissue culture dishes at a density of 10,000 to 15,000 cells per cm2 growth area for the CD105  and MSC-enriched cells and at a density of 10¨C15 x 105 cells per cm2 for the unfractionated MNCs. The medium was changed first after 72 hours and thereafter every 3 days. Between day 14 and day 16, the cells were detached by an incubation with 0.25% trypsin-EDTA, which lasted between 5 and 10 minutes, and were replated at a density ranging from 5,000 to 6,000 cells per cm2 for expansion. The cells were subcultured by trypsinization and replating when 90% confluence had been reached, after which they were assayed or stored in 85% complete medium (DMEM containing 10% FCS), 5% BSA, and 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in liquid N2 for future use.9 m3 |# N# Z* n7 x  y5 ?% E

CFU-F Assay. MNCs and CD105¨C and CD105  cells were separately plated onto six-well plates at 105 cells per well, and the media were changed as described in the previous section. Between 14 and 16 days later, the media were removed, and the cells were washed with 1x PBS. The cells were then fixed by incubation with 4% formaldehyde, stained with hematoxylin, and incubated at room temperature for 5 minutes. The wells were washed with tap water, and the plates were dried. Aggregates of 50 cells or more were scored as one colony-forming unit-fibroblast (CFU-F).; k' ]' T& `) _8 w- h

Flow Cytometry. Aliquots (0.5¨C1.5 x 106 cells) of fresh human BM-derived MNCs, freshly isolated CD105  cells, and culture-expanded CD105  cells were used separately for the analysis of cell-surface molecules. The cells were washed with 1x PBS, resuspended in fluorescence-activated cell sorter (FACS) buffer consisting of 2% BSA and 0.1% sodium azide (Sigma-Aldrich) in 1x PBS, and stained with fluorochrome-conjugated mouse anti-human CD105, CD31, CD106, CD90 (Ancell Corp., Bayport, MN, http://www.ancell.com), CD44, CD29, CD14, CD34, and CD45 (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) monoclonal antibodies according to the manufacturer¡¯s recommendations by using mouse monoclonal isotype antibodies (immunoglobulin  G1, IgG2) to detect any nonspecific fluorescence. The cells were washed with 1x PBS, resuspended in 0.5 ml of FACS buffer, and analyzed for the expression of the aforementioned human antigens by using FACScan and CellQuest software for data collection and analysis (both items from Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Gating was set up to 1% or less of isotype-stained cells (IgG1 or IgG2). In case more than one population was detected, a separate gating was set up to each population.0 x$ c0 c/ o8 f5 m+ Q) `+ k+ m; J0 h

In Vitro Differentiation Assays! h1 J8 n1 \+ w, Y

Osteogenesis Assay. To induce osteogenic differentiation in vitro, CD105  cells derived from BM were plated at a density of 3,000 cells per cm2 in DMEM containing 10% FCS (maintenance medium), as well as in an induction medium consisting of the maintenance medium plus 0.05 mM ascorbic acid-2-phosphate, 10 mM ß-glycerophosphate, and 0.1 µM dexamethasone (Decadron; Merck & Co., Whitehouse Station, NY, http://www.merck.com) (Sigma-Aldrich). At 1, 2, and 3 weeks after addition of the supplement, the cells were lysed with alkaline buffer solution (Sigma-Aldrich) containing 0.5% Triton X-100 and 10 mM MgCl2 (for the alkaline phosphatase  assay) or incubated with 0.5 N HCl solution (for the calcium deposition assay). For the ALP assay, the cell lysates were incubated with assay buffer containing 0.75 M 2-amino-2-methyl-1-propranolol (pH 10.3) for 10 minutes at 37¡ãC with p-nitrophenylphosphate as a substrate. For the calcium deposition assay, the cell lysates were incubated with gentle shaking for 24 hours at 4¡ãC. The samples were assayed for calcium content by using a calcium kit (Sigma-Aldrich). Protein content was measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, http://www.piercenet.com).
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Adipogenesis Assay. To induce adipogenic differentiation, CD105  cells derived from BM were plated in DMEM containing 10% FCS and grown until confluence. The medium was then replaced with high-glucose DMEM containing 10% FCS, 1 µM dexamethasone, 10 µM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 100 µM indomethacin (induction medium) or high-glucose DMEM containing 10% FCS and 10 µM insulin (maintenance medium). At confluence, cells assigned to the "adipo-differentiated" group were grown in induction medium for 3 days followed by 3 days of growth in maintenance medium (one cycle of induction and maintenance). These cells underwent three cycles of induction and maintenance before they were harvested. Cells assigned to the "adipo control" group were grown in maintenance medium for the entire period of differentiation. At the end of the assay, wells were stained using Oil Red O staining to confirm adipogenic differentiation." w2 `. Q* d% Z- A7 Y

Chondrogenesis Assay. To induce chondrogenic differentiation, aliquots of 2.5 x 105 CD105  hMSCs derived from BM were centrifuged in 15-ml conical centrifuge tubes and incubated overnight in DMEM containing 10% FCS. One day later, in "chondro control" cultures, this medium was replaced with medium containing 1% FCS; 6.25 µg/ml mixture of insulin, human transferrin, sodium selenite, bovine serum albumin, and linoleic acid; 50 nM ascorbic acid; and 0.1 µM dexamethasone (maintenance medium). In "chondro-differentiated" cell cultures, the original medium was replaced with an induction medium containing 10 ng/ml transforming growth factor-ß1 (TGF-ß1) (CytoLab Ltd., Rehovot, Israel, http://www.cytolab.com). Cell pellets in each group were kept in conical tubes in the incubator for 3 weeks, and then pellets were fixed with formalin, embedded in paraffin, and subjected to Alcian Blue staining to confirm chondrogenic differentiation.9 d" i, ?' g" H2 s

Infection of Noncultured CD105  Cells with Adeno-LacZ or Lenti-Green Fluorescent Protein. CD105  cells isolated from BM were resuspended in complete growth medium (DMEM containing 10% FCS) at 3¨C6 x 106 cells per ml in 15-ml conical tubes. The cells were mixed with recombinant human adenovirus type 5 encoding the ß-galactosidase gene, LacZ, under the cytomegalovirus promoter (adeno-LacZ, , a kind gift from Dr. M. Mezzina, Genethon, France), and incubated in 95% air/5% CO2 at 37¡ãC for 2 hours. After incubation, the cells were washed twice with 1x PBS and seeded onto six-well plates at 15,000 cells per cm2.

Implantation of DiI-Labeled CD105  Cells in an Ectopic Site. Bone marrow-derived hMSCs isolated using CD105 (from four independent donors) were immediately labeled with Vybrant DiI cell-labeling solution (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). Labeling was performed by resuspending the cells in serum-free DMEM at a concentration of 106 cells per ml, mixing the cell suspension with Vybrant DiI solution (15 µl/ml cell suspension), and incubating the cells in the dark for 30 minutes on a shaker in an atmosphere of 95% air/5% CO2 at 37¡ãC. DiI-labeled noncultured cells (1¨C1.5 x 106 cells per implant) were mixed with 5 µg of recombinant human BMP-2 (rhBMP-2), mounted on a 3 x 3 x 3-mm collagen sponge (DuraGen; Integra Lifesciences Corporation, NJ, http://www.integra-ls.com), and implanted subcutaneously into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice that were between 6 and 8 weeks of age (n = 4).8 R( f/ Y" ^5 ^. t+ w+ H6 v6 {
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Histology. For hematoxylin and eosin (H&E) staining, the implants were harvested 2 and 4 weeks postimplantation, fixed immediately by incubation in 4% formalin solution, and decalcified by incubation in 0.5 M EDTA in saline (pH 7.2). The tissues were dehydrated and embedded in paraffin and sectioned. To assess bone formation, H&E stain was applied to the slides.3 O3 ]+ l' p- T1 M; ]6 V

For fluorescent confocal microscopy, implants were fixed in 4% paraformaldehyde, embedded in ornithine carbamyl transferase compound, and frozen in liquid nitrogen, and frozen sections were made. DiI-labeled cells were visualized using fluorescent confocal microscope.2 k4 R5 S! M1 [# c3 j
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Micro-Computerized Tomography Study. A quantitative morphometric analysis of the bone mass was undertaken with the aid of micro-computerized tomography (µCT) scanning (micro-CT 40; Scanco Ltd.). New bone formation was measured using µCT three-dimensional reconstruction and an analysis of total bone volume (in cubic millimeters).3 X" \* O* J. m: `
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Statistical Analysis. Assays were performed using hMSCs obtained from at least three different donors. For each donor, the samples within each assay were assayed in three repeated experiments. Statistical tests for significance were performed using the Mann-Whitney test, and the minimal criterion for significance was determined to be a probability level less than .05. In the in vivo assays, four transplants of hMSCs from four different donors were used.
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RESULTS

Isolation of CD105  Cells from Human BM
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CD105 is a widely accepted marker used to detect the presence of hMSCs. Our intention was to verify the coexpression of CD105 with three other markers that might display a very low expression rate and are typical of hematopoietic cells: CD34, CD14, and CD45. Surface flow cytometric analysis of freshly separated human BM MNCs demonstrated that CD105 was expressed in 2.3% ¡À 0.45% of the MNCs (Table 1). Compared with isotype-stained MNCs (Fig. 1A), the hematopoietic markers that were coexpressed with CD105 were expressed at low levels (Fig. 1B¨C1D), with the exception of CD45, which was coexpressed with CD105 at approximately 60% of the CD105  fraction of MNCs. Most of the CD105  cells that expressed CD45 did so at low levels, however, as can be seen in the fluorescence levels detected by flow cytometry of cells stained with anti-CD105 and anti-CD45 antibodies (Fig. 1D). Once the MNCs had been isolated from whole BM, the cells were washed and resuspended in MACS buffer, and anti-CD105 antibodies conjugated to magnetic microbeads (CD105 microbeads) were added. To isolate the CD105  cells, we took advantage of the magnetic properties of the microbeads that were conjugated to the anti-CD105 antibody. Cells identified by this method as CD105  cells were analyzed for the expression of CD105 by performing flow cytometry, which demonstrated a purity of 79% ¡À 3.2% (Table 2; Fig. 1E). Surprisingly, only 36.1% ¡À 5.8% of the isolated CD105  cells expressed the CD45 antigen (Fig. 1F); 26.8% ¡À 5% of them expressed CD31 (Table 1; Fig. 1G) but did not express CD34 (Fig. 1H). Fresh noncultured CD105  cells were significantly smaller than culture-expanded CD105  hMSCs (Fig. 1I).  y; Y7 N2 O) @$ r5 j

Table 1. Analysis of MNCs and fresh CD105  cells

Figure 1. Immunophenotypes of bone marrow (BM)-derived mononuclear cells (MNCs) and freshly isolated CD105  cells. (A¨CI): Mononuclear cells were obtained from fresh human BM by separation on a density gradient centrifuge (A¨CD). CD105  cells were used for this analysis immediately following CD105 separation (E¨CI). Aliquots of 0.5¨C1.5 x 106 cells were stained with specified mouse anti-human antibodies (A¨CI) as described in Materials and Methods. The relative numbers of cells (shown as percentages) in the different quadrants were determined and are presented as means ¡À standard errors of the means (SEMs). The numbers displayed in the graphs in (E) through (I) indicate the percentage of stained cells for each specific antibody (empty portion of graph) compared with control isotype-stained cells (filled portion of graph). Cultured hMSCs (I, empty portion of graph) were trypsinized, washed, and analyzed for size (forward scatter), after which they were compared with fresh CD105  cells (I, filled portion of graph). Representative plots or graphs are shown. Numbers displayed in (D) and (E) indicate means ¡À SEMs (number of donors  4). Abbreviations: FL1-H, green color fluorescence; FL2-H, red color fluorescence.
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Table 2. Characterization of CD105  cells isolated from BM

Cultures containing CD105  cells that had been isolated from human BM MNCs were found to contain colonies of fibroblast-like cells 10¨C14 days after the initial plating; these colonies exhibited homogeneous morphological characteristics (Fig. 2A). Mononuclear cells from whole BM that did not undergo separation by CD105 microbeads contained a mixed population of fibroblast- and macrophage-like cells (Fig. 2B). A comparative analysis made using the CFU-F assay demonstrated that CD105  cells contained significantly higher levels of CFU-Fs (6.3 ¡À 1.4) than unseparated MNCs (1.1 ¡À 0.3), indicating that the CD105 separation enriched the MSC population (Table 2). It is important to note that the expression of CD14, the marker expressed on monocytes, on CD105  cells was very low. This indicates that macrophages are not isolated together with CD105  cells, even though in BM they are the hematopoietic cells with the highest ability to adhere to plastic (Fig. 1C). A marker flow cytometric analysis of culture-expanded hMSCs (CD105  hMSCs, passages 2¨C6) that had been isolated from BM by using CD105 microbeads showed that these cells displayed a positive reaction for CD106, CD105, CD29, CD44, and CD90 (Fig. 2C) and a negative reaction for CD14, CD34, CD31, and CD45 (Fig. 2D).0 a) R* ^. {- h3 M
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Figure 2. Morphological and flow cytometry analyses of culture-expanded CD105 isolated human mesenchymal stem cells. The cells were isolated from bone marrow (BM) by using a microbead-coupled antibody against CD105, after which they were plated and maintained in culture as indicated in Materials and Methods. (A, B): Photomicrographs showing CD105  cells (A) and unseparated MNCs (B) obtained after 10 days of culture. Original magnification x40. (C, D): Expression of surface molecules on culture-expanded BM-derived CD105  cells. Numbers displayed in the plots in (C) and (D) indicate the percentage of stained cells for each specific antibody (empty portion of graph) compared with control isotype-stained cells (filled portion of graph). Abbreviations: FL1-H, green color fluorescence; FL2-H, red color fluorescence.4 N* x/ p, E) p' b' X

In Vitro Differentiation of CD105  Cells

The goal of the next step was to characterize the osteogenic potential of the CD105  population in vitro. The osteogenic potentials of cell populations were tested after culture (10¨C14 days in culture) under conditions that induced the differentiation of MSCs into the osteogenic lineage . In the presence of ascorbate, dexamethasone, and ß-glycerophosphate, a significant increase in ALP activity was observed in CD105  hMSCs compared with the same type of cells cultured in the absence of these supplements (Fig. 3A). This result was observed throughout the entire experiment (3 weeks). Similar to the ALP activity, a continuous and significant increase in calcium deposition (expressed as an optical density of 575 nm) was observed during the differentiation period at 1, 2, and 3 weeks (Fig. 3B). To demonstrate more fully the in situ expression of ALP in CD105  hMSCs during osteogenic differentiation, ALP staining was performed (Fig. 3C). In another stage of the in vitro studies, we showed that culture-expanded CD105  cells can be induced to undergo both adipogenic (positive Oil Red O staining in Fig. 3D) and chondrogenic (positive Alcian Blue staining in Fig. 3E) differentiation.+ w, v7 T' `, M3 K

Figure 3. In vitro-expanded human mesenchymal stem cells (hMSCs) isolated with CD105 microbeads differentiate into osteogenic, adipogenic, and chondrogenic lineages in vitro. For the osteogenesis assay, bone marrow-derived CD105  cells expanded in culture (10¨C14 days in culture) were seeded onto 24-well plates at a density of 3,000 cells per cm2 and maintained in culture under conditions that would induce osteogenic differentiation. (A): Alkaline phosphatase activity was assessed in cell lysates 1, 2, and 3 weeks after the addition of an osteogenic supplement. The alkaline phosphatase (ALP) activity was assessed as the release of p-NP per minute normalized to total cell protein (in micrograms). (B): Calcium deposition was measured in cell lysates by examining the formation of the calcium-cresolphthalein complexone complex and expressed at an OD of 575 nm. Note the significant increases (*, p ' H" G6 z3 T+ Y5 C4 N

Noncultured CD105  Cells Engraft and Differentiate into Cartilage and Bone In Vivo9 t5 @% {2 k2 Y  t& ?6 s

To determine the ability of fresh noncultured CD105  cells to differentiate and form bone tissue in vivo, the cells were implanted into NOD/SCID mice. CD105  cells were able to respond to exogenously administered rhBMP-2, resulting in bone formation through the endochondral pathway .Noncultured CD105  cells (1¨C1.5 x 106 cells), which had been isolated by using CD105, were labeled with DiI, mixed with rhBMP-2, and placed under the skin of NOD/SCID mice using a collagen sponge as a carrier. Cartilage and bone formation were detected in the harvested implants 2 weeks after their placement, as observed following µCT scanning and staining with H&E (Fig. 4A, 4B, respectively). Micro-CT imaging demonstrated the formation of both cortical and trabecular bone within the implant (Fig. 4A). This imaging also demonstrated that the implant took the shape of the implanted collagen sponge. The bone volume measured at the implant site was 3.3 mm3.
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Figure 4. Engraftment and differentiation of noncultured human mesenchymal stem cells (hMSCs) implanted in an ectopic site. Noncultured hMSCs, which had been isolated from bone marrow samples, were labeled with the fluorescent cell tracer DiI and placed with recombinant human bone morphogenetic protein-2 under the skin of nonobese diabetic/severe combined immunodeficient mice. Two weeks later, the implants were harvested, fixed, and subjected to micro-computerized tomography (µCT) scanning followed by embedding in ornithine carbamyl transferase compound. (A): µCT scans of the harvested implant. (B, D): H&E staining of sections of the implants reveals newly formed cartilage and bone (B, arrowheads) located mainly in the periphery of the implant. Original magnifications x10 (B) and x40 (C, D). A magnification of the zone marked by the rectangle in (C) and (D) showed that hMSCs labeled with DiI were observed to have the morphological characteristics of chondrocytes (E, arrows) and osteoblasts (E, double arrows). Original magnification x100 (E). Abbreviations: 2D, two-dimensional; 3D, three-dimensional; BV, bone volume; CS, collagen sponge.# `% M6 b3 Q, @  h/ Z
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The region of newly formed bone was localized using both DiI and H&E staining. Photomicrographs of stained tissue showed the integration of implanted CD105  cells in the newly formed bone (Fig. 4C, 4D). Evident chondrocytes (Fig. 4E, arrows) and lining osteoblasts (Fig. 4E, double arrows) labeled with DiI were identified in the newly formed cartilage and bone tissue by performing confocal fluorescence microscopy.

Genetic Engineering of Noncultured CD105  Cells

Genetically modified hMSCs constitute an efficient and attractive tool for inducing tissue regeneration in skeletal tissues. Our main interest was to estimate whether noncultured CD105  cells could be genetically engineered before they were placed in culture, within a minimal time after their isolation. CD105  cells were isolated from BM samples by using CD105 microbeads and transfected in the first step with adeno-LacZ. After 10 days in culture, the resulting hMSC colonies were fixed and stained with 5-bromo-4-chloro-3-indolyl-ß-D-galactoside to estimate the expression of ß-galactosidase. Positively stained hMSCs were identified within these cultures at an estimated efficiency of infection of 10% (Fig. 5A). In addition, we tested the ability of lenti-GFP to transduce noncultured CD105  cells. Surprisingly, the efficiency of lenti-GFP transduction (44% GFP-positive cells) was much higher than that of adeno-LacZ transduction (Fig. 5B, 5C).

Figure 5. Noncultured human mesenchymal stem cells can be genetically engineered using adeno-LacZ and lenti-green fluorescent protein (GFP). Freshly isolated CD105  cells were infected with adeno-LacZ (A), not infected (mock infection) (B), or infected with lenti-GFP (C), as described in Materials and Methods, and either replated in culture plates until analyzed (A¨CC) or implanted 1 day following infection (D, E). Adeno-LacZ-infected cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-ß-D-galactoside reagent, and lenti-GFP-infected cells were analyzed using flow cytometry to measure the percentage of GFP  cells compared with mock-infected cells. (D, E): For the implantation procedure, the cells were loaded onto collagen sponges, implanted under the skin of nonobese diabetic/severe combined immunodeficient mice, and visualized macroscopically 3 days postimplantation by using a noninvasive fluorescent in vivo imaging system (D, arrowhead) or microscopically by using a fluorescent in vivo confocal microscopy system (CellVizio) (E, arrow). Abbreviations: FL1-H, green color fluorescence; M1, marker of gating.

To test the in vivo survival of CD105  cells that had been genetically engineered and implanted prior to culture, CD105  cells were transduced with lenti-GFP, and the cells were placed in culture medium overnight. The following day, these cells were implanted under the skin of NOD/SCID mice. Three days postimplantation, a noninvasive fluorescent in vivo imaging system was used for macrographic localization of the cells (Fig. 5D). At the single cell level, the cells were detected using a novel in vivo fibered confocal fluorescence microscopy system that enabled visualization of single GFP-positive cells within the implant (Fig. 5E).
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DISCUSSION

In this study, we demonstrated for the first time that hMSCs could be immunoisolated using CD105, genetically modified, and differentiated in vivo to form bone, without passing through the culture expansion stage. On the other hand, most previous studies involving hMSCs have described culture-expanded cells  and 3.6% CD49a  cells, respectively, in MNCs.0 L8 e: _8 @0 j! L
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We found that approximately 80% of CD105  fraction is positive for CD105 by flow cytometry. This may be explained by the fact that antibodies for immunoisolation and for flow cytometry are raised against different epitopes of CD105 antigen. In addition, this may be a result of the heterogenous expression of CD105 by hMSCs. Interestingly, we found that immunoisolated CD105  cells expressed lower levels of CD45 than CD105  cells within unseparated MNCs (demonstrated using flow cytometry .
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Our results demonstrate that the CD105 isolation method can enrich the hMSC population, as seen in the results of the CFU-F assay (Table 2). In addition, our results demonstrated that using the CD105-based immunoisolation we can obtain a homogenous population of hMSCs at both the morphological and immunophenotypic levels in a relatively minimal period of culture of 10 days after initial separation (Fig. 2). Similar to previous reports , we show that culture-expanded hMSCs isolated using CD105 microbeads display a positive reaction for CD105 (endoglin), CD29 (ß1 integrin), CD44 (hyaluronate), CD90 (Thy-1), and CD106 (VCAM-1) and that these cells express very low levels of the hematopoietic markers CD14 (macrophage marker), CD34 (hematopoietic stem cells), CD45 (leukocyte common antigen), and CD31 (endothelial marker); this indicates that these cells have the characteristics of hMSCs (Fig. 2). These findings demonstrate the reliability of CD105-based immunoisolation of hMSCs from BM that enable rapid purification of hMSCs population for in vivo application and for obtaining homogenous population for in vitro studies.

As shown by calcium deposition, ALP activity, and staining assays, CD105  cells were able to differentiate into osteoblastic cells in vitro, thus demonstrating that CD105  cells do have an osteogenic potential similar to that of previously described hMSCs . In addition, CD105  cells were able to differentiate into both adipogenic and chondrogenic lineages in vitro, indicating another characteristic of MSCs (Fig. 3).

The isolation and immediate transplantation of osteogenic cells for bone repair is a very promising strategy for the development of clinical platforms to regenerate and repair large bone defects. After we demonstrated the in vitro osteogenic potential of hMSCs that had been isolated by CD105 microbeads, our goals were to assess the in vivo osteogenic potential of fresh noncultured CD105  cells and to estimate the feasibility of gene transfer into these cells. In the first step, we tested the hypothesis that fresh noncultured CD105  cells were able to respond to rhBMP-2 in vivo and to differentiate into bone. We found that noncultured hMSCs within CD105  cells responded to the presence of rhBMP-2 in vivo by differentiating into cartilage and bone cells through the endochondral bone formation pathway, as shown in Figure 4. In addition, cell tracking showed that these fresh cells not only integrated into the newly formed bone but also survived for at least 2 weeks postimplantation, as shown in histological sections by using confocal microscopy. This finding supports the results of the clinical study reported by Horwitz et al. (1999), in which MSCs within fresh, unprocessed BM allografts gave rise to mature osteoblasts in the bones of recipients with osteogenesis imperfecta .) Z' B6 D# R) m+ d) Y" A

One novel approach to enhance the potency and differentiation potential of MSCs lies in the genetic modification of these cells to express a specific gene. Fresh noncultured CD105  cells could be efficiently transduced with reporter genes. The ability to achieve such a modification in stem cells without the need for a culturing stage would be of great advantage in the clinical settings. In our study, we hypothesized that freshly CD105  cells could be genetically modified to express a gene of interest. The transgene expression reached nearly 45% when using lentiviral vectors (Fig. 5); however, we believe that other viral vectors or nonviral methods may be more efficient than the adenovirus and lentivirus used in the current study.
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In relation to the clinical settings, the aim of the immunoisolation is to enrich implants such as bone substitutes with MSCs that provide osteogenic progenitors. Muschler et al. demonstrated an increase in fusion scores following enrichment of bone substitutes with bone marrow cells .4 A& W. R/ X) F1 x/ L+ v5 p

CONCLUSION
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In this report, we demonstrate that the CD105 antigen can be used to isolate an hMSC-enriched population from BM. The fresh CD105  hMSC population seems to have different phenotypic markers than culture-expanded hMSCs isolated using the same method. The fact that fresh noncultured hMSCs possess the ability to differentiate and integrate in vivo at sites of new bone formation indicates that these cells are promising candidates for in vivo bone regeneration and other future clinical applications.$ L, P; t  _" s7 Q
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DISCLOSURES
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS3 @% K/ O9 t3 z" B( y% {
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This study was funded in part by Consortium Bereshit for Cell Therapy, Ministry of Industry, Trade and Labor, Israel; and STEMGENOS (EU-FR5 Specific Targeted Research Project).
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