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. 2011 Mar;90(3):317–324. doi: 10.1177/0022034510387796

Mouse Mandible Contains Distinctive Mesenchymal Stem Cells

T Yamaza 1, G Ren 2, K Akiyama 1, C Chen 1, Y Shi 2, S Shi 1,*
PMCID: PMC3034836  NIHMSID: NIHMS260029  PMID: 21076121

Abstract

Although human orofacial bone-marrow-derived mesenchymal stem cells showed differentiation traits distinctly different from those of mesenchymal stem cells (MSCs) derived from long bone marrow (BMMSCs), mouse MSCs derived from orofacial bone have not been isolated due to technical difficulties, which in turn precludes the use of mouse models to study and cure orofacial diseases. In this study, we developed techniques to isolate and expand mouse orofacial bone/bone-marrow-derived MSCs (OMSCs) from mandibles and verified their MSC characteristics by single-colony formation, multi-lineage differentiation, and in vivo tissue regeneration. Activated T-lymphocytes impaired OMSCs via the Fas/Fas ligand pathway, as occurs in BMMSCs. Furthermore, we found that OMSCs are distinct from BMMSCs with respect to regulating T-lymphocyte survival and proliferation. Analysis of our data suggests that OMSCs are a unique population of MSCs and play an important role in systemic immunity. Abbreviations: BMMSC, bone marrow mesenchymal stem cell; HA/TCP, hydroxyapatite/tricalcium phosphate; OMSC, orofacial mesenchymal stem cell; OVX, ovariectomized.

Keywords: mesenchymal stem cells, mouse mandible, differentiation, tissue regeneration

Introduction

Bone marrow mesenchymal stem cells have been identified as a population of hierarchical post-natal stem cells with the potential to differentiate into various cell types such as osteoblasts, chondrocytes, and adipocytes (Friedenstein et al., 1974; Owen and Friedenstein, 1988; Beresford, 1989; Prockop, 1997; Azizi et al., 1998; Pittenger et al., 1999; Prockop et al., 2000; Toma et al., 2002; Gronthos et al., 2003). Since orofacial skeletal components such as tooth and jaw bones have been considered to be developed from migrating cranial neural crest cells (Chai et al., 2000), it is important to examine whether mouse orofacial bone/bone-marrow-derived MSCs (OMSCs) possess unique stem cell properties.

Human orofacial bone-marrow-derived MSCs have been shown to have characteristics distinct from those of human long-bone-marrow-derived MSCs (BMMSCs) in terms of differentiation traits (Matsubara et al., 2005; Akintoye et al., 2006), but the interplay of human orofacial bone-marrow-derived MSCs with immune cells, as seen in human BMMSCs, has not been explored. Mouse orofacial bone-marrow-derived MSCs have not yet been isolated, in part due to the small size and anatomically complicated structure of mouse jaw bones. In this study, we have developed a protocol to successfully isolate and expand mouse OMSCs. Also, we found that mouse OMSCs are distinct from mouse BMMSCs with respect to their interplay with T-cells.

Materials & Methods

Mice

Female C3H-strain (7- to 8 week-old) and C57BL/6-Tg(CAG-EGFP) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Immunocompromised beige nu/nu xid mice (8- to 10-week-old) were purchased from Harlan (Indianapolis, IN, USA). Ovariectomized C3H mice were analyzed at 4 wks post-surgery, and age-matched C3H mice were used as controls. Animal experiments in this study were performed under a research protocol approved by the Institutional Animal Care and Use Committee (University of Southern California, protocol #10874). All animals were maintained in a temperature-controlled room with a 12-hour alternating light-dark cycle and were fed sufficient diet and water ad libitum throughout the experimental period.

Antibodies

All primary antibodies used in this study are described in the Appendix.

Isolation of Mesenchymal Stem Cells (MSCs) from Mouse Jaw (mandibular) and Long Bones

We collected mandibular and long bones to isolate cells independently. The attached soft tissues and teeth, including incisors and molars, were removed from the bones. All nucleated cells (ANCs) from mandibular bones were obtained by digestion with 3 mg/mL collagenase type I (Worthington Biochem, Lakewood, NJ, USA) and 4 mg/mL dispase II (Roche Diagnostic, Indianapolis, IN, USA) for 60 min at 37°C. ANCs from long bones were obtained by flushing out from the bone marrow (Yamaza et al., 2008). Single-cell suspensions of ANCs from mandibular or long bones were obtained through 70-µm cell strainers (BD Bioscience, San Jose, CA, USA), and seeded at 1 to 1.5 x 106 or 10 to 15 x 106 on a 100-mm dish (Corning, Corning, NY, USA), respectively. MSCs were isolated and cultured following an established protocol (Yamaza et al., 2008). The cells formed single colonies, recognized as passage 0 (P0) MSCs, which were later collected and passed to P1 expansion for subsequent experiments. No feeder cells were used in MSC culture. Multiple colony MSCs derived from several mice (n = 3~5) were pooled and used in this study. Each test was repeated at least 3 times. To avoid hematopoietic cell contamination, we selected multiple numbers of single colony clusters and passed them to secondary culture.

Colony-forming Units–Fibroblastic (CFU-F) Assay

Independent isolated ANCs were seeded and cultured for CFU-F assay as described previously (Yamaza et al., 2008) and in the Appendix.

Population-doubling (PD) Assay

Independent isolated MSCs were used in this assay. The detailed methods are described in the Appendix.

Cell Proliferation Assay

P1 MSCs were assayed for bromodeoxyuridine (BrdU) incorporation as described in the Appendix.

Immunophenotype Analysis

P1 MSCs were stained for cell-surface markers and analyzed by flow cytometry (see Appendix). Positive cells were identified by comparison with the corresponding control cells in which a false-positive rate of less than 1% was accepted.

Multi-lineage Differentiation Assay

MSCs (P1) were cultured under in vitro osteogenic, adipogenic, and chondrogenic conditions as described in the Appendix.

MSC-mediated Tissue Regeneration in vivo

P1 BMMSCs or OMSCs (2 x 106 cells each), mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer, Warsaw, IN, USA), were implanted subcutaneously into immunocompromised mice, and analyzed as described in the Appendix.

Immunoblot Analysis

Immunoblotting was performed as described in the Appendix.

Isolation and Culture of Splenocytes and PanT-lymphocytes

Splenocytes and PanT cells were isolated and cultured (Appendix). The culture supernatant of PanT cells was enriched 10 times to use for conditioned media (CM).

Survival Assay for MSCs with Co-cultured PanT Cells

P1 MSCs were co-cultured with activated or naïve PanT cells under several conditions, and assayed by toluidine blue staining or apoptotic staining (see Appendix).

Cell Proliferation and Cell Death Assay of Splenocytes Co-cultured with MSCs

Splenocytes were co-cultured with P1 MSCs by means of the ChemoTx Chemotaxis System (NeuroProbe, Gaithersburg, MD, USA) (see details in the Appendix). Cell proliferation of activated splenocytes was analyzed by 3H-thymidine incorporation assay. DNA content in the cells was measured for cell death by flow cytometry.

Statistical Analysis

Each assay was repeated with 3 or 5 independent isolated cells and the results were averaged. Student’s t test was used to analyze significance between two groups. A P value of less than 0.05 was considered as a significant difference.

Results

Isolation and Characterization of Mouse OMSCs

Murine jaw bones contain a unique and complicated bone-bone marrow-tooth system (Appendix Fig. 1). To isolate OMSCs from mouse mandibles, we generated single-cell suspensions by enzyme digestion and plated them at a low density on plastic plates. OMSCs were capable of forming adherent clonogenic cell colonies originating from a single attached cell showing a typical fibroblast-like morphology (data not shown). These single colony clusters, termed colony-forming units-fibroblastic (CFU-F), were similar to primary cultured BMMSCs. However, OMSCs generated significantly higher numbers of CFU-F (55.33 ± 9.07 colonies per 1.5 x 106 cells/plate) than did BMMSCs (5.33 ± 0.58 per 1.5 x 106 cells/plate; P < 0.005) (Fig. 1A). In addition, OMSCs had a high number of population doublings and an elevated cell proliferation rate when compared with those of BMMSCs (Fig. 1B).

Figure 1.

Figure 1.

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Isolation and characterization of mouse OMSCs. (A) OMSCs generated higher numbers of CFU-F than did BMMSCs, as shown by toluidine blue staining. (B) The number of population doublings (PD) in OMSCs was higher than that in BMMSCs. (C,D) BrdU-positive (BrdU+) cells were significantly increased in OMSCs when compared with those in BMMSCs by BrdU incorporation assay (C). The proliferation rate was calculated as a percentage of BrdU-positive nuclei relative to the total nucleated cells (D). (E,F) Flow cytometry showed that OMSCs shared a surface molecule profile similar to that of BMMSCs. The results were representative of 5 (A-D) or 3 (E,F) independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.005. The graph bars show means ± SD.

Next, we performed flow cytometric analysis to examine the surface molecular expression in OMSCs (Figs. 1E, 1F). OMSCs failed to express hematopoietic markers (CD14, CD34, and CD45), but were positive for MSC-associated markers (CD73, CD105, CD106, SSEA-4, and Oct-4). It appears that OMSCs expressed significantly higher levels of SSEA-4 and Oct-4 when compared with BMMSCs. OMSCs were also highly positive for stem cell antigen 1 (Sca-1) and weakly positive for c-kit. Interestingly, OMSCs expressed the embryonic stem cell markers, stage-specific embryonic antigen 4 (SSEA-4) and Octamer 4 (Oct-4), two early stem cell markers previously found to be present in embryonic stem cells and BMMSCs (Izadpanah et al., 2006; Lamoury et al., 2006; Gang et al., 2007). The levels of expression of SSEA-4 (6.4%) and Oct-4 (6.0%) in OMSCs were higher than in BMMSCs (SSEA4, 4.2%; Oct-4, 2.6%), suggesting that OMSCs may contain a more primitive MSC subpopulation than BMMSCs.

Multi-lineage Differentiation of OMSCs

Under osteogenic culture conditions, OMSCs differentiated into osteoblasts and showed higher levels of alkaline phosphatase (ALP) activity (Fig. 2A), elevated capability of forming mineralized nodules (Fig. 2B), and higher levels of expression of osteoblastic markers, Runx2, ALP, and osteocalcin (OCN; Fig. 2C) when compared with BMMSCs. Moreover, OMSCs had a capacity to differentiate into Oil-red O-positive lipid-laden adipocytes and expressed adipogenic markers, lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor γ2 (PPARγ2). The adipogenic differentiation capacity of OMSCs was similar to that of BMMSCs (Figs. 2D, 2E). Alcian blue staining showed that OMSCs were capable of forming cartilage matrix in chondrogenic inductive aggregated cultures, as seen in BMMSCs (Figs. 2F, 2G). Also, safranin-O, toluidine blue, and anti-type II collagen antibody staining indicated that the capability of OMSCs to differentiate into chondrogenic cells is similar to that of BMMSCs (Appendix Fig. 2). When ex vivo-expanded OMSCs were transplanted into immunocompromised mice with hydroxyapatite/tricalcium phosphate (HA/TCP) as a carrier, OMSCs formed de novo bone structure on HA/TCP surfaces, as seen in BMMSC transplants (Fig. 2H). Interestingly, OMSCs generated a larger amount of bone tissue and fewer bone marrow elements than BMMSCs (Figs. 2I, 2J). In GFP mouse-derived OMSC transplants, we found both GFP-positive osteocytes and GFP-negative osteocytes (Figs. 2K, 2L), suggesting that donor OMSCs and recipient cellular components may contribute to new bone formation.

Figure 2.

Figure 2.

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Multi-lineage differentiation capacity of mouse OMSCs. (A-C) OMSCs showed an elevated osteogenic differentiation potential compared with BMMSCs. After 2 wks of osteogenic culture, OMSCs showed higher ALP activity than BMMSCs (A). After 6-week osteogenic induction, OMSCs formed significantly increased amounts of mineralized nodules than BMMSCs, as assessed by Alizarin red staining (B). The Alizarin-red-positive (Alizarin Red+) area was calculated as a percentage of total area (B). Immunoblot analysis revealed that OMSCs expressed higher levels of the osteoblastic-specific molecules Runx2, ALP, and OCN than BMMSCs at 2 wks post-induction (C). β-actin was used as an internal control. (D,E) OMSCs and BMMSCs possessed similar potential to differentiate into adipocytes at 4 wks post-adipogenic induction. Oil-red O staining showed that OMSCs are similar to BMMSCs in lipid accumulation. Numbers of Oil-red O-positive (Oil-Red-O+) cells were calculated as a percentage of total cells (D). Immunoblot assay indicated similar expression levels of adipocyte-specific molecules LPL and PPARγ2 in OMSCs and BMMSCs (E). (F,G) Alcian blue staining presented similar chondrogenic differentiation capacity in OMSCs and BMMSCs. Bar = 200 µm. (H-J) OMSCs were capable of forming de novo bone and bone marrow structures when transplanted subcutaneously into immunocompromised mice with HA/TCP as carrier. OMSCs formed a larger amount of bone matrix (B) than BMMSCs (H,I), but generated significantly fewer bone marrow (BM) elements than BMMSCs (J). Newly formed bone (I) and bone marrow (J) area were calculated as a percentage of total area. (K,L) Transplantation of GFP mouse-derived OMSCs. GFP-positive cells (open arrows) were detected in newly formed bone matrix, as well as GFP-negative cells (closed arrows), by immunohistochemistry with anti-GFP antibody (K). There were no GFP-positive cells when stained with control rabbit IgG antibody (L). CT, connective tissue; HA, HA/TCP carrier. Bar = 50 µm (H), 100 µm (K, L). The results were representative of 5 (A, B, D, F, G) or 3 (C, E, H-L) independent experiments. *P ≤ 0.05, ***P ≤ 0.005. The graph bars show means ± SD.

Interplay between OMSCs and T-lymphocytes

To examine whether T-lymphocytes affect OMSCs in vivo, we used ovariectomized (OVX) mice. Increased T-lymphocyte activation has been known to participate in bone loss in estrogen-deficient model OVX mice (Cenci et al., 2000; Roggia et al., 2001). It has been known that OVX caused a significant increase in the numbers of CFU-F in mouse BMMSCs, which may be a compensatory response to the reduced bone mineral density in OVX conditions (Miura et al., 2004). Here we found that OMSCs derived from OVX mice (OVX-OMSCs) showed a significantly increased number of CFU-F compared with OMSCs from sham mice (sham-OMSCs) (Figs. 3A, 3B). However, OVX-OMSCs showed similar cell proliferation rates (Figs. 3C, 3D) and a tendency to decreased osteogenic capability (Figs. 3E, 3F) compared with sham-OMSCs. RT-PCR and immunoblot analyses revealed that OVX-OMSCs showed partial deficiency of molecular expression in their osteogenic differentiation pathway (Appendix Figs. 3, 4).

Figure 3.

Figure 3.

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T-lymphocyte over-activated conditions in OVX mice led to OMSC impairment. (A,B) OVX mouse-derived OMSCs (OVX-OMSCs) and BMMSCs (OVX-BMMSCs) formed significantly high numbers of CFU-F when compared with sham mouse-derived OMSCs (Sham-OMSCs) and BMMSCs (Sham-BMMSCs), respectively. (C,D) BrdU incorporation assay revealed that OVX-BMMSCs showed higher proliferation rates than sham-BMMSCs. However, statistically, there was no significant difference between OVX-OMSC and the sham-OMSC group. OVX-OMSCs showed higher proliferation rates than OVX-BMMSCs. (E,F) After 6 wks of culture in osteogenic conditions, OVX-BMMSCs showed significantly decreased capacity of forming mineralized nodules when compared with sham-BMMSCs by Alizarin red staining. However, statistically, there was no significant difference between the OVX-OMSC and sham-OMSC groups. OVX-OMSCs showed higher mineralized nodule-forming capacity than OVX-BMMSCs. The results were representative of 5 independent experiments. *P ≤ 0.05, ***P ≤ 0.005. The graph bars show means ± SD.

Our previous report indicated that activated T-cells induce apoptosis of BMMSCs through the Fas/FasL pathway (Yamaza et al., 2008). To determine whether activated T-cells also directly impinge on OMSCs, we co-cultured OMSCs with PanT-lymphocytes. Although naïve PanT cells were unable to induce OMSC death, PanT cells activated by anti-CD3 antibody caused OMSC death, but not completely (Fig. 4A). When OMSCs were separated from PanT cells in a transwell co-cultured system, OMSCs failed to show cell death (Fig. 4A), suggesting that direct cell-cell contact is required to induce OMSC death by activated T-lymphocytes. Conditioned medium collected from activated PanT cells did not cause significant cell death in OMSCs (Fig. 4B), and neutralization with a cocktail of anti-tumor necrosis factor α (TNFα) and anti-interferon γ (IFNγ) antibodies failed to prevent OMSC apoptosis (Fig. 4B), indicating that soluble factors released from activated T-lymphocytes did not contribute to OMSC death. Furthermore, treatment with anti-Fas ligand (FasL) antibody or brefeldin A blocked OMSC death (Fig. 4C). In contrast, concanamycin A did not induce OMSC apoptosis (Fig. 4C), indicating that the Fas/FasL pathway, but not the perforine/granzyme pathway, contributes to the apoptosis of OMSCs. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining was used to confirm that OMSC death was due to apoptosis (Fig. 4D). Interestingly, we found that activated T-cells induced a greater cell death in BMMSCs than in OMSCs (Fig. 4D), which might be associated with the lower level of expression of Fas in OMSCs compared with that in BMMSCs, by immunoblot analysis (Figs. 4E, 4F).

Figure 4.

Figure 4.

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Mouse OMSCs interplay with T-lymphocytes. (A) PanT cells (PanT) activated by antibody against CD3 (anti-CD3Ab, 1 µg/mL) were capable of inducing significant cell death in OMSCs and BMMSCs, as shown by toluidine blue staining, but OMSCs appeared to experience less cell death than BMMSCs. Activated PanT cells failed to induce BMMSC and OMSC death when indirectly co-cultured by the Transwell system. (B) PanT culture-derived conditioned medium (CM) did not induce OMSC and BMMSC death. Neutralization with anti-IFNγ and anti-TNFα antibodies (1 µg/mL each) did not inhibit OMSC and BMMSC death. (C) Treatment with anti-FasL antibody (1 µg/mL) and brefeldin A (BFA, 10 mM), but not concanamycin A (ConA, 100 nM), blocked OMSC and BMMSC death. (D) The TUNEL-positive OMSC apoptosis rate was significantly less than the rate of BMMSC apoptosis. The percentage of TUNEL-positive (TUNEL+) nuclei was calculated compared with the total number of MSCs. (E) OMSCs expressed a significantly lower level of Fas compared with that in BMMSCs, by immunoblotting. (F) Cell proliferation of activated splenocytes was measured under BMMSC- or OMSC-co-culture system by 3H-thymidine incorporation assay. OMSCs suppressed splenocyte proliferation more effectively than BMMSCs. Anti-IFNγ antibody (25 µg/mL) or 1400W (0.2 mM) treatment showed partial inhibition of splenocyte proliferation in the OMSC or BMMSC co-culture system. (G) Naïve splenocytes were co-cultured with OMSCs or BMMSCs at different ratios for 60 hrs, and stained with propidium iodide for measurement of DNA contents by flow cytometry. OMSCs were more effective in inhibiting spontaneous apoptosis of naïve splenocytes compared with BMMSCs. Numbers on the histograms show averaged percentiles of cells containing hypodiploid DNA contents. The results are representative of 5 (A-D) or 3 (E-G) independent experiments. *P ≤ 0.05, ***P ≤ 0.005. The graph bars or values show means ± SD.

OMSCs Modulate Cell Survival and Cell Proliferation of T-lymphocytes

Our recent study indicated that the immunosuppressive property of BMMSCs was due to a high level of nitric oxide (NO) production induced by IFNγ via inducible nitric oxide synthase (iNOS; Ren et al., 2008). To compare the immunosuppressive capacity of OMSCs with that of BMMSCs, we co-cultured anti-CD3 antibody-activated splenocytes with different ratios of OMSCs and BMMSCs in the presence or absence of anti-IFNγ antibody or the iNOS inhibitor, 1400W. When the MSC-to-splenocyte ratio was 1:80, significant inhibition of T-cell proliferation was observed in the OMSC group, but not in the BMMSC group (Fig. 4G), suggesting that OMSCs had a stronger immunosuppressive effect than BMMSCs. This may be associated with the fact that OMSCs produced more NO than BMMSCs when treated with IFNγ and TNFα (Appendix Fig. 5). However, the inhibition effect of OMSCs was only partially blocked by anti-IFNγ antibody and iNOS inhibitor, indicating that other factor(s) might contribute to NO-mediated immunosuppression in OMSCs. Our previous studies demonstrated that BMMSCs are effective in preventing apoptosis of splenocytes in the absence of T-cell activation signals (Ren et al., 2008). Herein, when the MSC-to-splenocyte ratio was 1:20, 12% of naïve splenocytes underwent apoptosis in OMSCs cultures compared with 24% apoptotic naïve splenocytes in BMMSC culture (Fig. 4H), suggesting that OMSCs were more effective in maintaining naïve splenocyte viability than BMMSCs.

Discussion

Embryologic development and amalgamations of the complex array of bones and cartilage in the craniofacial region have revealed that the molecular mechanisms controlling skeletogenesis in the orofacial bones are unique and different from that in the axial and appendicular bones (Helms and Schneider, 2003). The discrepancy in bone development between orofacial bones and long axial/appendicular bones give rises to specific diseases in the orofacial bone region, such as periodontitis, cherubism (Ueki et al., 2001), and hyperparathyroid jaw tumor syndrome (Simonds et al., 2002), which affect only the bones of the jaw. Therefore, it is not surprising to find that human OMSCs are distinct from BMMSCs in terms of differentiation traits (Matsubara et al., 2005; Akintoye et al., 2006). In this study, we successfully isolated MSCs from mouse mandibular bones, OMSCs, and demonstrated their unique differentiation capacities and immunomodulatory properties, suggesting a potential use for a variety of mouse models to study dental and orofacial diseases.

Since mouse jaw bones contain limited quantities of marrow components, it is difficult to collect and expand MSC from the jaw bone marrow. Therefore, we used an enzyme digestion method to isolate MSCs from mandibular bones, and verified their MSC properties by clonogenic, multi-lineage differentiation, and tissue regeneration assays in comparison with mouse BMMSCs. It was reported that bone and bone marrow contain MSCs with similar stem cell properties, but slightly different in terms of surface molecule expression (Sanchez-Guijo et al., 2009). It is likely that OMSCs contain post-migrating cranial neural crest cells (Chung et al., 2009). Analogous to BMMSCs, OMSCs are a mixed MSC population containing cells derived from mandibular bone and bone marrow.

Ex vivo-expanded BMMSCs are capable of suppressing T-lymphocyte activity in vitro, which provides a foundation for the use of BMMSC transplantation to treat T-cell-associated disorders, such as acute graft-vs.-host-disease (GvHD) in mice and humans (Koc et al., 2002; Bartholomew et al., 2002; Deng et al., 2004; Xu et al., 2007; Ren et al., 2008). In contrast, activated T-lymphocytes can induce apoptosis of BMMSCs through the Fas/FasL pathway (Yamaza et al., 2008). Analysis of our data suggests that OVX-induced T-lymphocyte activation may contribute to OMSC differentiation deficiency. However, we cannot exclude other factors that may also contribute to OMSC deficiency in OVX mice. In general, the immunomodulatory property of MSCs may be associated with a high level NO production induced by IFNγ via enhanced iNOS expression in BMMSCs (Ren et al., 2008). In this study, we found that mouse OMSCs showed a stronger suppressive effect on the proliferation of anti-CD3 antibody-activated T-cells, along with high levels of NO production, when stimulated with IFNγ. We also found that OMSCs were capable of maintaining naïve splenocyte, including T-cell, survival more effectively than BMMSCs. Therefore, it is necessary to continue elucidating the underlying mechanisms of the interplay between OMSCs and immunity in various established mouse models.

Mouse OMSCs presented higher potency for osteoblastic differentiation than mouse BMMSCs, implying a site-specific bone-regenerating capacity, as seen in human OMSCs (Matsubara et al., 2005; Akintoye et al., 2006). Previously, SSEA-4 and Oct-4 were identified as embryonic stem cell markers, but recent studies have shown that human and rodent BMMSCs (Izadpanah et al., 2006; Lamoury et al., 2006; Gang et al., 2007) express SSEA-4 and Oct-4. It appears that these embryonic stem cell markers are expressed in early progenitors of BMMSCs. We found that OMSCs express significantly higher levels of SSEA-4 and Oct-4 than BMMSCs, suggesting that mouse OMSCs may contain more primitive stem cell progenitors.

Supplementary Material

Appendix
supp_90_3_317__index.html (573B, html)

Acknowledgments

The authors thank Dr. Larry Fisher for providing antibodies for this study. This work was supported by grants from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services (ARRA 1R01DE019413 to S.S. and Y.S., and R01DE017449 to S.S.), National Institutes of Health (AI057596 to Y.S.), and New Jersey Commission on Science and Technology (NJCST-2042–014–84 to Y.S.).

Footnotes

A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.

References

  1. Akintoye SO, Lam T, Shi S, Brahim J, Collins MT, Robey PG. (2006). Skeletal site-specific characterization of orofacial and iliac crest human bone marrow stromal cells in [the] same individuals. Bone 38:758-768 [DOI] [PubMed] [Google Scholar]
  2. Azizi SA, Stokes D, Augelli BJ, DiGirolamo C, Prockop DJ. (1998). Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats–similarities to astrocyte grafts. Proc Natl Acad Sci USA 95:3908-3913 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, et al. (2002). Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30:42-48 [DOI] [PubMed] [Google Scholar]
  4. Beresford JN. (1989). Osteogenic stem cells and the stromal system of bone and marrow. Clin Orthop Relat Res 240:270-280 [PubMed] [Google Scholar]
  5. Cenci S, Weitzmann MN, Roggia C, Namba N, Novack D, Woodring J, et al. (2000). Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-alpha. J Clin Invest 106:1229-1237 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chai Y, Jiang X, Ito Y, Bringas P, Jr, Han J, Rowitch DH, et al. (2000). Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development 127:1671-1679 [DOI] [PubMed] [Google Scholar]
  7. Chung IH, Yamaza T, Zhao H, Choung PH, Shi S, Chai Y. (2009). Stem cell property of postmigratory cranial crest cells and their utility in alveolar bone regeneration and tooth development. Stem Cells 27:866-877 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deng W, Han Q, Liao L, Li C, Ge W, Zhao Z, et al. (2004). Allogeneic bone marrow-derived flk-1+Sca-1-mesenchymal stem cells leads [sic] to stable mixed chimerism and donor-specific tolerance. Exp Hematol 32:861-867 [DOI] [PubMed] [Google Scholar]
  9. Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV. (1974). Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 17:331-340 [DOI] [PubMed] [Google Scholar]
  10. Gang EJ, Bosnakovski D, Figueiredo CA, Visser JW, Perlingeiro RC. (2007). SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 109:1743-1751 [DOI] [PubMed] [Google Scholar]
  11. Gronthos S, Zannettino AC, Hay SJ, Shi S, Graves SE, Kortesidis A, et al. (2003). Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci 116(Pt 9):1827-1835 [DOI] [PubMed] [Google Scholar]
  12. Helms JA, Schneider RA. (2003). Cranial skeletal biology. Nature 423:326-331 [DOI] [PubMed] [Google Scholar]
  13. Izadpanah R, Trygg C, Patel B, Kriedt C, Dufour J, Gimble JM, et al. (2006). Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 99:1285-1297 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W. (2002). Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 30:215-222 [DOI] [PubMed] [Google Scholar]
  15. Lamoury FM, Croitoru-Lamoury J, Brew BJ. (2006). Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct-4 and Rex-1. Cytotherapy 8:228-242 [DOI] [PubMed] [Google Scholar]
  16. Matsubara T, Suardita K, Ishii M, Sugiyama M, Igarashi A, Oda R, et al. (2005). Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells. J Bone Miner Res 20:399-409 [DOI] [PubMed] [Google Scholar]
  17. Miura M, Chen X-D, Allen MR, Bi Y, Gronthos S, Seo BM, et al. (2004). A crucial role of Caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J Clin Invest 114:1704-1713 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Owen M, Friedenstein AJ. (1988). Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 136:42-60 [DOI] [PubMed] [Google Scholar]
  19. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284:143-147 [DOI] [PubMed] [Google Scholar]
  20. Prockop DJ. (1997). Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71-74 [DOI] [PubMed] [Google Scholar]
  21. Prockop DJ, Azizi SA, Phinney DG, Kopen GC, Schwarz EJ. (2000). Potential use of marrow stromal cells as therapeutic vectors for diseases of the central nervous system. Prog Brain Res 128:293-297 [DOI] [PubMed] [Google Scholar]
  22. Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Roberts AI, et al. (2008). Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2:141-150 [DOI] [PubMed] [Google Scholar]
  23. Roggia C, Gao Y, Cenci S, Weitzmann MN, Toraldo G, Isaia G, et al. (2001). Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo. Proc Natl Acad Sci USA 98:13960-13965 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sanchez-Guijo FM, Blanco JF, Cruz G, Muntion S, Gomez M, Carrancio S, et al. (2009). Multiparametric comparison of mesenchymal stromal cells obtained from trabecular bone by using a novel isolation method with those obtained by iliac crest aspiration from the same subjects. Cell Tissue Res 336:501-507 [DOI] [PubMed] [Google Scholar]
  25. Simonds WF, James-Newton LA, Agarwal SK, Yang B, Skarulis MC, Hendy GN, et al. (2002). Familial isolated hyperparathyroidism: clinical and genetic characteristics of 36 kindreds. Medicine (Baltimore) 81:1-26 [DOI] [PubMed] [Google Scholar]
  26. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. (2002). Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 105:93-98 [DOI] [PubMed] [Google Scholar]
  27. Ueki Y, Tiziani V, Santanna C, Fukai N, Maulik C, Garfinkle J, et al. (2001). Mutations in the gene encoding c-Ab1-binding protein SH3BP2 cause cherubism. Nat Genet 28:125-126 [DOI] [PubMed] [Google Scholar]
  28. Xu G, Zhang L, Ren G, Yuan Z, Zhang Y, Zhao RC, et al. (2007). Immunosuppressive properties of cloned bone marrow mesenchymal stem cells. Cell Res 17:240-248 [DOI] [PubMed] [Google Scholar]
  29. Yamaza T, Miura Y, Bi Y, Liu Y, Akiyama K, Sonoyama W, et al. (2008). Pharmacologic stem cell based intervention as a new approach to osteoporosis treatment in rodents. PLoS ONE 3:e2615. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Appendix
supp_90_3_317__index.html (573B, html)
supp_0022034510387796_DS_10.1177_0022034510387796.pdf (743.6KB, pdf)

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