TISSUE-SPECIFIC STEM CELLS Mesenchymal Stem Cell-Organized Bone Marrow Elements: An Alternative Hematopoietic Progenitor Resource YASUO MIURA,a ZHIGANG GAO,b MASAKO MIURA,a BYOUNG-MOO SEO,a WATARU SONOYAMA,a WANJUN CHEN,a STAN GRONTHOS,c LI ZHANG,d SONGTAO SHIa a
National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland, USA; bDivision of Immunology/ Hematology, The Sidney-Kimmel Oncology Center, Johns Hopkins School of Medicine, Baltimore, Maryland, USA; c Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia; dDepartment of Physiology, The University of Maryland School of Medicine, Rockville, Maryland, USA Key Words. Mesenchymal stem cell • Hematopoietic stem cell • Platelet-derived growth factor • Bone marrow organ system
ABSTRACT Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent postnatal stem cells that have been used for the treatment of bone defects and graft-versus-host diseases in clinics. In this study, we found that subcutaneously transplanted human BMMSCs are capable of organizing hematopoietic progenitors of recipient origin. These hematopoietic cells expressed multiple lineages of hematopoietic cell associated markers and were able to rescue lethally irradiated mice, with successful engraftment in the recipient, sug-
gesting a potential bone marrow (BM) resource for stem cell therapies. Furthermore, we found that platelet-derived growth factor (PDGF) promotes the formation of BMMSCgenerated BM niches through upregulation of -catenin, implying that the PDGF pathway contributes to the formation of ectopic BM. These results indicate that the BMMSCorganized BM niche system represents a unique hematopoietic progenitor resource possessing potential clinical value. STEM CELLS 2006;24:2428 –2436
INTRODUCTION
used to treat a variety of disorders, including bone fracture [8], severe aplastic anemia [9], and acute graft-versus-host-disease [10]. The mechanism of BMMSC-mediated therapies may be attributable to either their osteogenic differentiation to form new bone or their modulating affects on immune cell responses [11]. BMMSCs are capable of forming ectopic bone and associated BM structures when transplanted into immunocompromised mice subcutaneously using hydroxyapatite tricalcium phosphate (HA/TCP) as a carrier vehicle [1, 12–14]. However, the functional role of BMMSC-organized BM structures is unknown. Our hypothesis is that BMMSC-organized BM niches contain HSCs that can be used for potential clinical therapies.
Bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of differentiating into a variety of cell types, including osteoblasts, chondrocytes, adipocytes, tendon cells, muscle cells, and neural cells [1–3]. The most encouraged differentiation trait of BMMSCs is osteoblastic differentiation in vitro and bone formation in vivo. Osteoblasts are responsible not only for forming bone in the normal adult bone remodeling process but also providing a specific niche microenvironment for hematopoietic stem cells (HSCs) governed by bone morphogenetic protein, parathyroid hormone, and Tie2/Angiopoietin-1 signaling pathways [4 – 6]. This emerging evidence suggest a new functional role of osteogenic cells for controlling HSC niches in vivo [1]. Inside the bone marrow (BM) niche microenvironment, interplay between BMMSCs and HSCs may determine homeostasis of BM environment. HSCs are the only stem cell population that is routinely used for human disease treatment [7]. On the other hand, human BMMSCs have been successfully
MATERIALS
AND
METHODS
Human BMMSC Culture Human BM cells were purchased from commercially available resources (AllCells LLC, Berkeley, CA, http://www.allcells.
Correspondence: Songtao Shi, D.D.S., Ph.D., Center for Craniofacial Molecular Biology, University of Southern California School of Dentistry, 2250 Alcazar Street, CSA 103, Los Angeles, California 90033, USA. Telephone: 323-442-3038; Fax: 323-442-2981; E-mail:
[email protected]; or Li Zhang, Ph.D., Center for Vascular and Inflammatory Diseases, Department of Physiology, University of Maryland School of Medicine, 800 West Baltimore Street, Baltimore, Maryland 21201, USA. Telephone: 410-7068040; Fax: 410-706-8121; E-mail:
[email protected] Received February 14, 2006; accepted for publication July 3, 2006; first published online in STEM CELLS EXPRESS July 13, 2006. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/ stemcells.2006-0089
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Miura, Gao, Miura et al. com). To isolate purified human BMMSCs and to exclude potential hematopoietic cell contamination, we used STRO-1 and MUC18 as cell surface markers to sort mononuclear cells from freshly collected bone marrow (bone marrow mononuclear cells [BMMNCs]) and subsequently expanded them in the culture to enrich cell numbers as reported previously [15]. The adherent cells were cultured in ␣-minimal essential medium (␣MEM) (Gibco, Carlsbad, CA, http://www.invitrogen.com) supplemented with 15% fetal bovine serum (FBS) (EquitechBio Inc., Kerrville, TX, http://www.equitech-bio.com), 100 M ascorbic acid 2-phosphate (Wako Chemical, Tokyo, Japan, http://www.wako-chem.co.jp/english), 2 mM L-glutamine (Biosource, Carlsbad, CA, http://www.invitrogen.com), 100 U/ml penicillin, and 100 g/ml streptomycin sulfate (Biosource). Prior to further experiments, the ex vivo expanded cells were applied to fluorescence-activated cell sorting (FACS) analysis to confirm the exclusion of hematopoietic cell contamination and the expression of BMMSC markers by the use of surface antigens CD14, CD34, CD45, CD44, CD166, C105, CD106, and CD90 [16].
Antibodies Rabbit anti-bone sialoprotein (anti-BSP) (LF-120) antibody (Ab) was provided by Dr. Larry Fisher (National Institute of Dental and Craniofacial Research (NIDCR), NIH, Bethesda, MD) [17, 18]. Rabbit anti--catenin Ab and mouse anti--actin Ab were purchased from Sigma-Aldrich (St. Louis, http://www. sigmaaldrich.com). Rabbit anti-N-cadherin Ab was purchased from Immuno-Biological Laboratories Co. (Takasaki, Gunma, Japan, http://www.ibl-japan.co.jp). Mouse anti-bromodeoxyuridine (anti-BrdU) Ab was from Zymed (Carlsbad, CA, http:// www.invitrogen.com). Rabbit anti-c-kit Ab and anti-plateletderived growth factor receptor- (anti-PDGFR) Ab were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www. scbt.com). The following Abs were obtained from Becton, Dickinson and Company (Franklin Lakes, NJ, http://www.bd.com): mouse monoclonal antibody (mAb) against GFP; rat mAb against the B220 and TER-119 antigens; phycoerythrin (PE)conjugated rat mAbs against Sca-1, CD45, CD18, CD71, CD3, CD4, IgM, CD19, Gr-1, CD11b, CD41, and TER-119 molecules; and PE-conjugated isotype-matched control antibodies.
BMMSC Transplantation
Human BMMSCs (2.0 – 4.0 ⫻ 106) at passage 3 were mixed with 40 mg of HA/TCP ceramic powder (Zimmer Inc., Warsaw, IN, http://www.zimmer.com) at 37°C rotated for 90 minutes and then transplanted subcutaneously into the dorsal surface of 8-week-old female immunocompromised beige nude mice (NIH-bg-nu/-xid; Harlan Sprague Dawley, Inc., Indianapolis, IN, http://www.harlan.com) as previously described [13, 14, 18]. These procedures were performed in accordance with specifications of approved animal protocol (NIDCR 04-317). In some experiments, the same number of primary gingival fibroblasts, dental pulp stem cells (DPSCs) [18] and mouse primary osteoblasts (POBs) [19] were used. The transplants were harvested at 8 weeks post-BMMSC transplantation unless mentioned specifically and were applied to further experiments. www.StemCells.com
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Cocultures of BMMNCs with BMMSCs Human BMMSCs were cultured in a 75-cm2 culture flask under an osteogenic inductive condition for the indicated period as previously described [18]. Calcium accumulation on BMMSCs was confirmed by Alizarin red staining. Human BMMNCs (2.8 ⫻ 106) isolated from the same individual as a BMMSC donor were directly added on the osteogenic differentiated BMMSCs monolayer in 10 ml of serum-free StemSpan medium supplemented with 100 ng/ml kit ligand, 100 ng/ml Flt-3 ligand, and 100 ng/ml thrombopoietin (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). The cultures were maintained for 1 week. Cells cocultured with BMMSCs were collected by pipetting the cell suspension and applied to numeration of all nucleated cells (ANCs) and colony-forming cell (CFC) assays.
Enhanced Green Fluorescent Protein Labeling A lentivirus vector containing enhanced green fluorescent protein (eGFP) construct was used in this study [20]. The cells isolated from the BMMSC transplants were resuspended at 1–2 ⫻ 105 cells per milliliter in a serum-free StemSpan medium supplemented with 100 ng/ml kit ligand, 100 ng/ml Flt-3 ligand, and 20 ng/ml thrombopoietin (KFT medium; R&D Systems Inc., Minneapolis, http://www.rndsystems.com). For transduction, viral supernatant (titers, 2– 8 ⫻ 106) in QBSF-60 medium (Quality Biological Inc., Gaithersburg, MD, http://www. qualitybiological.com) were mixed at a 1:1 ratio with KFT medium containing transplant-derived cells in the presence of 8 g/ml polybrene. The transduction mixture was centrifuged at 1800g for 4 hours at 32°C–35°C. After this spinoculation procedure, the cells were washed once and cultured in KFT medium. The transduction procedure was repeated the following day before being transplanted. The eGFP-labeled cells were applied to further experiments.
Engraftment of BMMSC Transplant-Derived Cells BMMSC transplants were harvested aseptically from the dorsal surface of immunocompromised mice at 8 weeks post-transplantation and kept in phosphate-buffered saline containing 2% FBS (PBS/FBS). The BM cells including hematopoietic and mesenchymal cells were retrieved from the transplants by physical separation of HA/TCP particles using a razor blade. The cells were washed twice using PBS/FBS and resuspended in PBS/FBS. Single-cell suspension was obtained by passing the cells through a 40-m strainer. The number of 5.5 ⫻ 106 cells derived from 10 BMMSC transplants was administered intravenously through tail veins into C57Bl/6 mice (n ⫽ 7; Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) that had received total body irradiation (TBI) of 11 Gy at a dose rate of 0.83 Gy/minute from cesium-137 gamma rays prior to the transplantation. For the positive (n ⫽ 3) and negative (n ⫽ 4) control experiments, BM cells from the long bones of immunocompromised mice and cells isolated from the DPSC transplants were administered into lethally irradiated mice, respectively. Survival of mice was inspected daily for at least the observed period of 180 days. To examine engraftment of transplanted cells, the cells isolated from BMMSC transplant were labeled with eGFP by the use of lentivirus vector system, and 1 ⫻ 105 eGFP-labeled cells were administered intravenously into sublethally irradiated (TBI of 7.5 Gy) C57Bl/6 mice. Engraftment
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was examined by immunohistochemical and FACS analyses (Becton, Dickinson and Company) at 8 –12 weeks postinfusion.
Homing of eGFP-Positive BM Cells Immunocompromised mice carrying the BMMSC transplants received daily i.v. injection of 64 mg/kg cyclophosphamide (CY) (Sigma-Aldrich) dissolved in PBS for 4 days. BM cells (1.5 ⫻ 107 ANCs per mouse) isolated from long bones of eGFP transgenic mice (Jackson Laboratory) were administered intravenously through the tail vein into the CY-preconditioned immunocompromised mice. Homing of eGFP-positive BM cells into the BMMSC-transplants or long bones of the recipient mice was examined at 8 weeks postinjection by immunohistochemical and FACS analyses.
Small Interfering RNA Transfection
BMMSCs (2 ⫻ 105) were plated in a six-well tissue culture plate in 2 ml of ␣MEM supplemented with 10% FBS, 100 M ascorbic acid 2-phosphate, 2 mM L-glutamine for 1–2 days until the cells reached 60%– 80% confluence. Human PDGFR small interfering (si)RNA (double-stranded [ds]DNA) and negative control siRNA were purchased from Santa Cruz Biotechnologies Inc. Transfection of the siRNAs was performed according to the manufacturer’s instruction. Briefly, the siRNA in transfection medium (Santa Cruz Biotechnologies Inc.) at concentration of 53 nM was incubated with BMMSCs for 6 hours at 37°C in a CO2 incubator. After additional 24 hours regular medium culture, BMMSCs were harvested for analysis.
Histological Examination The BMMSC transplants harvested at the indicated period after transplantation were fixed with 4% paraformaldehyde, decalcified with 10% EDTA (pH 8.0), and embedded in paraffin. Sections were deparaffinized and stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, sections were incubated with primary antibodies at 1:40 –1:200 dilutions for 1 hour. Isotype-matched negative control antibodies (Zymed) were used under the same conditions. The broad-spectrum immunoperoxidase AEC kit (Picture Plus; Zymed) was subsequently used to detect the proteins according to the manufacturer’s instruction. The sections were stained with hematoxylin for counter staining. BrdU-positive cells were detected by the use of detection kit (Zymed) according to the manufacturer’s instructions. BrdU-long-term retaining (LTR) assays were performed as previously described [4]. Tartrate-resistant acid phosphate (TRAP) staining was performed according to the previous report [21]. Quantification of the size of BM niche and number of hematopoietic cells were calculated on more than 15 representative areas at ⫻100 magnification with previously described method [22]. The size of BM niche was defined as the BM areas surrounded by the mononuclear spindle-shaped osteoblasts lining on the bone surfaces by using NIH image software (NIH Image, Rockville, MD) [4, 22]. The number of hematopoietic cells was determined by counting the nucleated cells in the BM niche area. Experiments of double immunofluorescence were performed as previously described with a modification [23].
FACS Analysis
Cells (1 ⫻ 106) isolated from the BMMSC transplants and long bones of mice were incubated with 1 g/ml of PE-conjugated
mAbs for 45 minutes at 4°C. PE-conjugated isotype-matched IgG was used as a control. After being washed three times with PBS containing 2% FBS, the cells were applied to FACS analysis.
Western Blotting Analysis
BMMSCs were treated with 1 g/ml PDGF-BB (Sigma-Aldrich) for the indicated periods. The expression of -catenin, N-cadherin, and -actin was examined by Western blotting analysis as previously described [19].
CFC Assays Cells isolated from BMMSC transplants or cells cocultured with BMMSCs were applied to CFC assays using the methylcellulose-based medium (MethoCult GF M3444 for mouse cells, GF H4434 for human cells; Stem Cell Technologies) according to the manufacturer’s instructions. Briefly, the number of 1 ⫻ 104 cells were resuspended with 100 l of Iscove’s modified Dulbecco’s medium ⫹ 2% FBS (Stem Cell Technologies) and mixed with 1 ml of methylcellulose-based medium followed by seeding cells on a 35-mm dish. The number of colonies was counted 12–14 days after the incubation at 37°C.
Statistical Analysis Statistical significance was analyzed by Student’s t test between two parameters. Overall survival was calculated starting from the irradiation date to the mice death or to the end of the observation. Survival curve was analyzed by the Kaplan-Meier method and compared by the log-rank test. A p value of less than .05 was considered statistically significant.
RESULTS To investigate BMMSC-mediated hematopoiesis, purified human BMMSCs were transplanted into the dorsal surface of immunocompromised mice using HA/TCP as a carrier. Bone formation was initiated at 4 weeks post-transplantation along with a significant in-growth of blood vessels in the interstitial compartments (Fig. 1A). In the BMMSC transplants, BM structure emerged only around established bone usually at 6 – 8 weeks post-transplantation. At 6 weeks post-transplantation of BMMSCs, enlarged blood vessels were found in replacement of the connective tissue and might associate with osteoblasts, suggesting a potential communication between vascular endothelial cells and osteoblasts for subsequent BM formation (Fig. 1B). Furthermore, hematopoietic cells in the newly formed BM were closely associated with the osteoblasts (Fig. 1C), which mimicked a physiological setting analogous to regular BM. In contrast, primary fibroblasts failed to generate any new bone and BM under the same condition of BMMSCs (Fig. 1D), indicating that the recipient microenvironment responded specifically to the BMMSC-mediated osteogenesis [13, 14, 17]. Immunohistochemical analysis on the BMMSC transplants revealed that the B220 antigen, a marker for lymphoid cell lineage, was positive for the cells in the BM of the BMMSC transplants (Fig. 1E, 1F) and that the pattern of staining was similar to that in the BM of long bones (Fig. 1G, 1H). The TER-119 antigen, an erythroid cell marker, was also found to be positive in BM cells in the BMMSC transplants (Fig. 1I). The presence of osteoclasts, known to originate from myeloid progenitor cells, were found on the surface of bones inside the
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Figure 1. BMMSC-mediated BM formation. (A): Four weeks post-transplantation. Initiation of osteogenesis (open arrows) was found on the surface of HA. In-growth of BV was noted in the IC. (B): Six weeks post-transplantation. Osteoblasts (open arrows) on the surface of bone were associated with endothelial cells of BV that contained R. (C): Eight weeks post-transplantation. Newly formed bone was integrated with HA, and BM was closely associated with osteoblasts (open arrows). (D): Primary gingival fibroblasts were transplanted with HA as a negative control. Only CT was found. (E–H): Immunohistochemical analysis using anti-B220 antibody (Ab) showed lymphoid lineage cells in the BM cells of BMMSC transplants (E) and of long bones. (G): Immunohistochemical analysis using control IgG showed negative staining in BMMSC transplants (F) and in long bones (H). (I): Immunohistochemical analysis using anti-TER-119 Ab showed erythroid lineage cells in the transplants. (J): Osteoclasts on the surface of the bones showed a positive tartrate-resistant acid phosphate (TRAP) staining (arrows) in 8-week BMMSC transplants. (K): There were no TRAPpositive cells in IC in 2-week BMMSC transplant. (L): Megakaryocytes (yellow arrows) were noted in the BMMSC transplants. (M): Cells isolated from BMMSC transplants formed hematopoietic colonies in the culture of methylcellulose-based medium. No colony was detected in cultured cells isolated from the DPSC transplants under the same conditions. Abbreviations: B, bone; BM, bone marrow; BMMSC, bone marrow-derived mesenchymal stem cell; BV, blood vessel; CFC, colony-forming cell; CT, connective tissue; DPSC, dental pulp stem cell; HA, hydroxyapatite tricalcium phosphate particles; IC, interstitial compartment; R, red blood cells.
BMMSC transplants at 8 weeks post-transplantation as assessed by TRAP staining (Fig. 1J). There were no TRAP-positive cells in the BMMSC transplants prior to the BM formation at 2 weeks post-BMMSC transplantation (Fig. 1K). The presence of megakaryocytes (Fig. 1L) and hematopoietic CFCs [13, 14] in BMMSC transplants (Fig. 1M) further revealed the existence of hematopoietic progenitors in the BM of the BMMSC transplants. However, another mesenchymal stem cell population, DPSCs [17, 18], transplants failed to form CFCs (Fig. 1M), providing further evidence of unique hematopoiesis-supportive properties of BMMSCs. We further characterized hematopoietic cells in the BMMSC transplants by the use of FACS analysis. We harvested cells from either BMMSC transplants or long bones in the recipient mice of the BMMSC transplants and found that the cells isolated from BMMSC transplants showed the same expression pattern of various lineages of hematopoietic cell surface markers including www.StemCells.com
CD45 (pan-leukocytes), CD18 (leukocytes), CD19 (B lymphocytes), CD4 (T lymphocytes), Gr-1 (granulocytes), CD41 (megakaryocytes), TER-119 (erythroid cells) and Sca-1 (HSCs) as that from long bones in the recipient mice (Fig. 2A; Table 1). More importantly, C57BL/6 mice that received lethal TBI and then following systemic administration of BM cells isolated from the BMMSC transplants survived for at least the observed period of 180 days, similar to lethally irradiated mice that received transplantation of BM cells isolated from long bones of the BMMSC transplanted mice (Fig. 2B). Conversely, all mice that received transplantation of cells isolated from the DPSC transplants died from day 8 to day 12 after TBI (Fig. 2B). To confirm engraftment of the donor-derived cells in the recipient mice, the cells isolated from BMMSC transplants were labeled with eGFP using a lentivirus vector system. The eGFP-labeled cells were administered intravenously through the tail vein into the sublethally irradiated
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Table 1. Expression of hematopoietic cell markers
CD45 Sca-1 c-kit CD34 CD18 CD4 CD19 CD11b Gr-1 CD71 TER-119 CD41
Transplant BM cells (%)
Long bone BM cells (%)
74.8 9.22 10.5 2.51 76.5 ND 6.33 59.2 49.9 32.1 35.0 6.68
71.4 12.7 7.44 1.34 73.3 ND 6.44 58.8 44.2 32.7 30.9 4.58
Fluorescence-activated cell sorting analysis showed that BM cells isolated from the bone marrow-derived mesenchymal stem cells (BMMSCs) transplants (transplant BM cells) and from the long bones of the BMMSC recipient mice (long bone BM cells) had similar expression patterns. Representative data from 13 different samples are shown here. Abbreviations: ND, not detected; BM, bone marrow.
Figure 2. BM cells in the BMMSC transplants contain functional hematopoietic cells. (A): Characterization of BM cells isolated from BMMSC transplants. Cells isolated from either the BMMSC transplants or the long bones of the BMMSC transplant recipient mice were harvested and applied to fluorescence-activated cell sorting analysis. Both cells showed similar expression patterns, which are typically found in hematopoietic cells. (B): Engraftment of BM cells isolated from BMMSC transplants. Cells isolated from either the BMMSC transplants (red line) or the long bones of the BMMSC transplant recipient mice (blue line) were administered intravenously into C57Bl/6 mice that had received total body irradiation of 11 Gy prior to the administration. Lethally irradiated mice were rescued by systemic transplantation of cells isolated from BMMSC transplants (red line) and BM cells from long bones (blue line) as a positive control. Cells isolated from of the same number of DPSC transplants (green line) were used as a negative control to show a nonrescue result. Survival of mice was observed for 180 days as demonstrated with the Kaplan-Meier survival curve. Abbreviations: BM, bone marrow; BMMSC, bone marrow-derived mesenchymal stem cell; DPSC, dental pulp stem cell.
C57BL/6 mice. Furthermore, FACS analysis revealed that the eGFP-labeled transplanted cells engrafted in the long bones of recipient mice expressed hematopoietic cell marker CD45, CD44, or CD71 (data not shown). These findings indicated that the BM cells isolated from BMMSC transplants contain functional hematopoietic progenitor cells to engraft and support hematopoiesis in recipients. We further explored the potential that human BMMSCmediated transplants serve as a bone marrow organ system (BMOS) that possess both functional hematopoietic progenitor/ stem cells and niche microenvironment for the hematopoietic cells. To test this possibility, we assessed whether BMMSCs
were responsible for orchestrating the formation of the niche microenvironment. The osteoblasts derived from BMMSCs were located on the surface of newly formed bones in the BMMSC-transplants and were confirmed to express an osteogenic marker, BSP (Fig. 3A). We found that they expressed newly identified HSC niche-associated molecules, -catenin and N-cadherin [4, 5] (Fig. 3B–3D). Recent study has demonstrated that BrdU-LTR HSCs reside on the niches around the trabecular areas in the lone bones of mice [4]. We used the same strategy to examine whether ectopically generated BMMSCtransplants contain the HSC niches. Immunohistochemical analysis showed that BrdU-positive cells were found in the hematopoietic compartment along the surface of bones in the BMMSC transplants (Fig. 3E), and they expressed c-kit, a HSC marker (Fig. 3F). Previous data have shown that mature normal human trabecular bone cells form abundant bone but do not support hematopoietic marrow formation upon in vivo transplantation in immunocompromised mice (A. Majolagbe and P.G. Robey, unpublished data). In this study, human BMMSCs cultured under osteogenic-inductive conditions in vitro for 2 weeks (Fig. 3K) and POBs harvested from mouse calvariae demonstrated a osteogenesis but lacked formation of BM elements (Fig. 3G, 3H). This evidence suggests that a trait of osteogenic differentiation of BMMSCs may possess a superior capacity to induce hematopoiesis compared with other osteogenic cells. To further explore the capacity of BMMSCs in hematopoiesis, we cocultured BMMNCs with undifferentiated (Fig. 3I), 1-week osteogenic differentiated (Fig. 3J), or 4-week osteogenic differentiated (Fig. 3L) BMMSCs in vitro. The number of CFCs and ANCs was decreased in the coculture condition with osteogenic differentiated BMMSCs compared with that with undifferentiated BMMSCs (Fig. 3M, 3N), further supporting the finding that osteogenic differentiation of BMMSCs altered their interactive capacity with hematopoietic cells, probably through modulating BM niches. Taken together, BMMSCmediated osteogenesis is a commanding process that occurred prior to hematopoiesis for the BMOS formation.
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Figure 3. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) organizes BM niche formation. (A): Expression of the osteogenic marker bone sialoprotein (BSP) (open arrows) in osteoblasts on the surface of bone in the BMMSC transplants. Endothelial cells of BV in the BM structure were negative for BSP (closed arrows). (B–D): Expression of BM niche markers. The osteoblasts showed a positive staining for -catenin (B) and N-cadherin (C) in the BMMSC transplants. Rabbit control IgG was used as a negative control to verify specific staining of the antibodies (D). (E): Bromodeoxyuridine-long-term retaining (BrdU-LTR) cells (yellow arrows) in BM of the BMMSC transplants resided along with osteogenic cells (blue arrow) on the surface of bone. (F): BrdU-LTR cells (left, red) co-expressed c-kit (right, merged yellow), a maker for HSCs. (G, H): In vitro osteogenic differentiated human BMMSCs (G) and mouse primary osteoblasts (H) were capable of forming bone in the transplants but lacked significant BM structure at 8 weeks post-transplantation. (I–L): Alizarin red staining showed development of calcium accumulation (arrows) in human BMMSCs under osteogenic inductive condition in vitro for 0 weeks (undifferentiated) (I), 1 week (J), 2 weeks (K), and 4 weeks (L). (M, N): Cocultures of BMMNCs with osteogenic differentiated BMMSCs. The number of hematopoietic CFCs (M) and ANCs (N) were decreased when BMMNCs were cocultured with 1-week or 4-week osteogenic differentiated BMMSCs. Before starting coculture of BM cells with osteogenic BMMSCs, undifferentiated BMMSCs were osteogenic induced for either 1 week or 4 weeks in an osteogenic induction medium. Then, BM cells were added onto 1-week osteogenic inducted BMMSCs (1-week point) or 4-week osteogenic inducted BMMSCs (4-week point). The medium for coculture of BM cells with osteogenic differentiated BMMSCs was a serum-free StemSpan medium supplemented with 100 ng/ml kit ligand, 100 ng/ml Flt-3 ligand, and 100 ng/ml thrombopoietin. The period of coculture was 1 week under both coculture conditions, and then BM cells were collected for colony assay. Abbreviations: ANCs, all nucleated cells; B, bone; BM, bone marrow; BV, blood vessel; CFC, colony-forming cell; CT, connective tissue; HA, hydroxyapatite tricalcium phosphate particles.
Next, we assessed the functional role of BM niches in the BMMSC transplants/BMOSs. HSCs have to specifically reach the BM niche microenvironment through the systemic circulation, which is referred to homing. We injected eGFP-BM cells from the long bones of the transgenic mice into CY-preconditioned recipient immunocompromised mice carrying BMMSC transplants. FACS analysis showed that cells isolated from the BMMSC transplants contained eGFP-positive cells and that they expressed multiple hematopoietic cell markers, including Sca-1, CD71, CD19, IgM, CD3, and CD11b (Fig. 4A). A similar cell population was found in the BM cells isolated from long bones of the recipient mice (Fig. 4A). Immunohistochemical analysis further showed that eGFP-positive cells homed to the BMMSC-organized BM structure as well as to BM in long bones of the recipient mice at 8 –12 weeks after the transplantation (Fig. 4 B– 4E). These results indicate that BM niches organized by BMMSCs were capable of attracting and fostering www.StemCells.com
BM cells serving as functional homing niches, analogous to the regular BM. For the development of practical applications of the BMMSC transplants/BMOSs, it is important to generate BM niches that contain sufficient numbers of hematopoietic cells. When BMMSCs were treated with PDGF-BB and then transplanted into immunocompromised mice, the size of the BM niches and the number of hematopoietic cells was significantly increased compared with nontreated BMMSC transplants (Fig. 5A). In vitro experiments showed that PDGF promoted upregulation of -catenin and N-cadherin in the BMMSCs (Fig. 5B). The functional role of PDGF-BB in the BM niche formation was further verified by a siRNA experiment. When the expression of PDGFR was inhibited by siRNA treatment in BMMSCs (Fig. 5C), bone and BM formation were found to be impaired when the siRNA-treated BMMSCs were transplanted into immunocompromised mice subcutaneously (Fig. 5D, 5E).
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Figure 4. Functional role of BM niches in the bone marrow-derived mesenchymal stem cell (BMMSC) transplants. (A): Homing of BM cells to the BMMSC-organized BM niches. Fluorescence-activated cell sorting analysis of the cells isolated from the BMMSC transplant showed homing of eGFP-positive cells that expressed hematopoietic cell associated markers (green squares), Sca-1 (hematopoietic stem cells), CD71 (erythroid cells), CD19 (B lymphocytes), IgM (B lymphocytes), CD3 (T lymphocytes), and CD11b (myeloid cells). BM cells isolated from the long bones of eGFP BM-transplanted (normal BM) and untransplanted (control) mice were used as positive and negative controls, respectively. (B–E): Homing of BM cells to the BMMSC-organized BM niches. Immunohistochemical analysis using anti-GFP monoclonal antibody demonstrated that eGFP-positive BM cells (yellow arrows) homed to the BM compartment in the BMMSC transplants (B) and of long bones (D) in the immunocompromised recipient mice. Immunostaining with control IgG showed negative staining of the BMMSC transplants (C) and the long bones (E). Abbreviations: B, bone; BM, bone marrow; eGFP, enhanced green fluorescent protein.
DISCUSSION Given the significant role attributed to osteoblasts in maintaining and supporting the HSC niches in the BM [1, 4, 5], it is conceivable that BMMSCs, which are known osteoblastic progenitors, may also play a vital role in the organization of the HSC niches. Early histological studies have suggested that ex vivo expanded human BMMSCs are capable of generating bone and associated BM structures upon subcutaneous transplantation in vivo [1, 14, 15, 17]. However, there is no evidence to demonstrate whether the newly formed BM structures contain functional hematopoietic cells. Our study clearly demonstrated the presence of multiple lineages of hematopoietic cells, including myeloid, lymphoid, erythroid, and hematopoietic progenitor cells in the BMMSC transplants based on immunohistological
Mesenchymal Stem Cells and Bone Marrow and FACS analysis. More importantly, we observed that mice that had received lethal TBI could be rescued by systemic transplantation of BM cells isolated from the BMMSC transplants. These findings indicate that the BM cells isolated from the BMMSC transplants contain functional hematopoietic progenitor cells to engraft and support hematopoiesis in recipients. As well as a potential capacity as a resource for HSCs, we demonstrated that the BMMSC transplants maintain HSCs niche microenvironments. In the BMMSC transplants, osteoblasts derived from BMMSCs expressed newly identified HSC nicheassociated markers N-cadherin and -catenin and colocalized with BrdU-LTR HSCs, a primitive subpopulation of HSCs. The formation of HSC niches in the BMMSC transplants was functionally confirmed by the observation that systemically administered allogeneic BM cells homed to the BMMSC transplants and reconstituted donor-derived hematopoiesis in which hematopoietic cells expressing Sca-1, a marker for hematopoietic stem cells, and multiple lineage specific hematopoietic associated markers were detected. These findings indicate that BMMSC transplants serve as a BMOS that contains both functional HSC niches and BM cells. To date, several resources are clinically available for HSC transplant, including BM stem cells, peripheral blood stem cells, and cord blood stem cells. It is conceivable that the generation of BMOSs that contains functional HSCs and BM cells may have a unique impact on clinical stem cell therapy. For the development of practical applications of the BMMSC transplants/BMOSs, it is important to generate BM niches that contain sufficient numbers of hematopoietic cells. In this regard, we used PDGF-BB since it was reported that PDGF stimulates the growth of STRO-1-positive BM stromal cells in vitro [24]. When BMMSCs were treated with PDGF-BB and then transplanted into immunocompromised mice, the size of the BM niches and the number of hematopoietic cells were significantly increased compared with nontreated BMMSC transplants. The mechanism of PDGF-promoted BM niche formation may result from PDGF-mediated upregulation of -catenin and N-cadherin in BMMSCs because the interaction between -catenin and N-cadherin has been identified as a major regulatory factor for maintaining BM niches [4]. The reason for a slightly upregulated expression of -catenin and N-cadherin in control group without PDGF may be the stimulation of multiple growth factors in FBS. When PDGF signaling was inhibited in BMMSCs by PDGFR siRNA, osteogenesis and hematopoiesis were blocked in the BMMSC transplants, signifying the important role of the PDGF pathway during the bone and BM formation. In this study, we provided experimental evidence to identify BMMSCs as belonging to a population of postnatal stem cells that are capable of organizing a BMOS that contains functional hematopoietic progenitors and the niche microenvironment. The generation of BMMSC-mediated BMOSs represents a unique model to study how BMMSCs regulate hematogenesis/osteogenesis in vivo and a unique approach to transplant hematopoietic system in the recipients.
ACKNOWLEDGMENTS We gratefully acknowledge Cun-Yu Wang and Carolyn Coppe for discussions and critical reading of the manuscript and Tina
Miura, Gao, Miura et al.
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Figure 5. Involvement of PDGF in the BM niche formation. (A): Bone marrow-derived mesenchymal stem cells (BMMSCs) cotransplanted with PDGF-BB (⫹PDGF) organized more BM niches and higher numbers of hematopoietic cells than did untreated BMMSCs (⫺PDGF). (B): Western blotting analysis showed an elevated expression of -catenin and N-cadherin in the in vitro cultured BMMSCs following the treatment of PDGF-BB (⫹PDGF) for the indicated time compared with the untreated BMMSCs (⫺PDGF). The -actin was used as protein loading control. (C): Western blotting analysis showed an inhibition of PDGFR expression in BMMSCs transfected with siRNA targeting PDGFR. (D, E): siRNA transfection targeting PDGFR blocked bone and BM formation in 8-week BMMSC transplants (D) compared with the control siRNA-transfected BMMSC transplants (E). Abbreviations: B, bone; BM, bone marrow; BV, blood vessel; HA, hydroxyapatite tricalcium phosphate particles; IC, interstitial compartment; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; siRNA, small interfering RNA.
Kilts and Driss Ehirchiou for animal technical support. This work was supported by the intramural program of NationalInstitute of Dental and Craniofacial Research, NIH, Department of Health and Human Services, and in part by NIH Grant R01-HL61589 from the National Heart, Lung, and Blood Institute (to L.Z.). Z.G. is currently affiliated with the Neuro-
REFERENCES 1
Friedenstein AJ, Chailakhyan RK, Latsinik NV et al. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974;17:331–340.
2
Owen M, Friedenstein AJ. Stromal stem cells: Marrow-derived osteogenic precursors. Ciba Found Symp 1988;136:42– 60.
3
Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–74.
Oncology Branch, National Cancer Institute, NIH, Bethesda, MD.
DISCLOSURES The authors indicate no potential conflicts of interest.
11 Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105:1815–1822. 12 Umezawa A, Maruyama T, Segawa K et al. Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo. J Cell Physiol 1992; 151:197–205. 13 Krebsbach PH, Kuznetsov SA, Satomura K et al. Bone formation in vivo: Comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts. Transplantation 1997;63:1059 –1069. 14 Kuznetsov SA, Krebsbach PH, Satomura K et al. Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo. J Bone Miner Res 1997;12:1335–1347.
4
Zhang J, Niu C, Ye L et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425:836 – 841.
5
Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841– 846.
15 Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003; 18:696 –704.
6
Arai F, Hirao A, Ohmura M et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118:149 –161.
16 Miura Y, Miura M, Gronthos S et al. Defective osteogenesis of the stromal stem cells predisposes CD18-null mice to osteogenesis. Proc Natl Acad Sci U S A 2005;102:14022–14027.
7
Korbling M, Estrov Z. Adult stem cells for tissue repair—A new therapeutic concept? N Engl J Med 2003;349:570 –582.
17 Batouli S, Miura M, Brahim J et al. Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J Dent Res 2003;82:976 –981.
8
Quarto R, Mastrogiacomo M, Cancedda R et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 2001;344:385–386.
18 Gronthos S, Mankani M, Brahim J et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–13630.
9
Fouillard L, Bensidhoum M, Bories D et al. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia 2003;17:474–476.
19 Miura M, Chen XD, Allen MR et al. A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J Clin Invest 2004;114:1704 –1713.
10 Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439 –1441.
20 Gao Z, Golob J, Tanavde VM et al. High levels of transgene expression following transduction of long-term NOD/SCID-repopulating human cells with a modified lentiviral vector. STEM CELLS 2001;19:247–259.
www.StemCells.com
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Mesenchymal Stem Cells and Bone Marrow
21 Chiang CY, Kyritsis G, Graves DT et al. Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide. Infect Immun 1999;67:4231–4236.
23 Seo BM, Miura M, Gronthos S et al. Multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149 –155.
22 Shi S, Gronthos S, Chen S et al. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol 2002;20:587–591.
24 Gronthos S, Simmons PJ. The growth factor requirements of STRO-1positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 1995;85:929 –940.
