Immunomodulatory properties of stem cells from human exfoliated deciduous teeth

1Human bone marrow mesenchymal stem cells (BMMSCs) have been identified as a population of postnatal stem cells with the potential to self-renew and differentiate into osteoblasts, chondrocytes, adipocytes, and neural cells. BMMSCs also exhibit immunomodulatory and regulatory effects on T and B lymphocytes, dendritic cells, and natural killer cells, indicating an attractive feature for cell therapy. In addition, culture expanded BMMSCs may fail to express MHC-class II antigens on their surfaces, therefore allogenic BMMSCs have been used in treating a variety of diseases such as acute graft-versus-host-disease (GVHD), ameliorating Hematopoietic Stem Cell engraftment, and systemic lupus erythematosus (SLE). Recently, mesenchymal stem cells derived from other tissues have also been found to possess immunomodulatory functions which offer opportunities to find more effective and feasible mesenchymal stem cell sources for cell therapies.

2Stem cells from human exfoliated deciduous teeth (SHED) have been isolated from naturally exfoliated deciduous teeth with the capacity to differentiate into osteogenic and odontogenic cells, adipocytes, and neural cells. As neural crest cell-associated postnatal stem cells, SHED express a variety of neural cell markers including nestin, beta III tubulin, GAD, NeuN, GFAP, NFM, and CNPase. Also, SHED are able to form bone when transplanted in vivo and offer obvious bone regeneration for repairing calvarial defects in a mouse model. It is unknown whether SHED possess immunomodulatory function as seen in BMMSCs. In this study, we compare immuno-regulatory properties between SHED and BMMSCs and utilize SHED transplantation to treat SLE-like diseases in a murine model.

3C57BL/6J and C3MRL-Faslpr/J (MRL/lpr) mice (female, six- to seven-week-old) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Beige nude/nude Xid (III) mice (female, 8- to 12-week-old) were purchased from Harlan (Indianapolis, IN, USA). All animal experiments were performed under an institutionally approved protocol for the use of animal research (University of Southern California protocol #10874 and #10941).

4Human exfoliated deciduous incisors were obtained as discarded biological samples from children (six- to eight-year-old) at the Dental Clinic of the University of Southern California following the approved Institutional Review Board guidelines. Healthy bone marrow aspirates from iliac bone and peripheral blood mononuclear cells (PBMNCs) of healthy volunteers were purchased from AllCells (Berkeley, CA, USA).

5Mononuclear cells isolated from the remnant dental pulp tissue of the deciduous incisors were cultured as reported previously. BMMSCs culture was described previously. The detailed protocols were described in Additional file 1.

6The procedure for single colored flow cytometry (FCM) was performed as described previously [, and Additional file 1]. The samples were analyzed on a FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA). Some cells were used for immunoblot analysis and immunofluorescent staining.

7CFU-F assay was performed according to a previous study [, and Additional file 1].

8The proliferation of each MSC population was performed by bromodeoxyuridine (BrdU) incorporation assay as previously described [ and Additional file 1].

9Telomerase activity was evaluated by telomeric repeat amplification protocol (TRAP) assay using real-time polymerase chain reaction (PCR) [, and Additional file 1].

10Osteogenic differentiation assays of SHED and BMMSCs were performed according to previous publications. Osteogenic markers and mineralized nodule formation were assessed as described previously [ and Additional file 1].

11Adipogenic assay in vitro of each stem cell population was performed as described previously [, and Additional file 1].

12Xenogeneic transplantation was performed using immunocompromised mice as described. Each MSC population was subcutaneously transplanted into beige nude/nude Xid (III) mice using hydroxyapatite tricalcium phosphate (HA/TCP) as a carrier. Eight weeks post-transplantation, the transplants were harvested for histological analysis. Detail methods were described in the Additional file 1.

13Ten μg total protein was loaded and analyzed by immunoblotting as previously described [, and Additional file 1].

14PBMNCs or T cells were co-cultured with or without SHED or BMMSCs under several culture conditions as described in Additional file 1. Cell death analysis and induction of Tregs and Th17 cells were described in Additional file 1

15Under general anesthesia, SHED or BMMSCs (1 × 105 cells/10 g body weight in 100 μl PBS) were infused into MRL/lpr mice via tail vein at 16 weeks (n = 3) according to previous study. MRL/lpr mice (16-week-old) received physiological saline (n = 3) were used as experimentally control mice. All mice were sacrificed at 20 weeks of age, and from them were collected peripheral blood, kidney, and long bones (femur and tibiae).

16Flow cytometric staining and analysis were performed as previously reported [, and Additional file 1].

17Several biomarkers, including anti-dsDNA antibody and anti-nuclear antibody ANA, complement 3 (C3), interleukin 6 (IL6), IL10, IL17, soluble receptor activator for nuclear factor κB ligand (sRANKL), and C-terminal telopeptides of type I collagen (CTX), creatinine, urine protein in biofluid samples (peripheral blood serum and urine) were measured by enzyme linked immunosorbent assay (ELISA) [, and Additional file 1].

18Kidneys and long bones (femurs) harvested from mice were fixed and processed to make paraffin sections. The sections were used for further experiments [Additional file 1].

19Histomorphometric analysis was quantified as described previously. Detailed methods were described in Additional file 1.

20All data are expressed as the mean ± SD of, at least, triplicate determinations. Statistical difference between the values was examined by Student's t-test. The P values less than 0.05 were considered significant.

21All primary antibodies used in this study were described in Additional file 1 and listed on Table S1 in Additional file 1. All primer pairs were listed in Table S2 in Additional file 1

22Although SHED are capable of differentiating into a variety of cell types, their detailed mesenchymal stem cell properties remain to be elucidated. Herein, we used flow cytometry, immunoblot analysis, and immunocytostaining analysis to demonstrate that SHED at passage 3 expressed many mesenchymal surface markers, including STRO-1, SSEA4, CD73, CD105, CD146, and CD166 but were negative for CD34 and CD45 (Figures 1A-C). In comparison to BMMSCs, SHED expressed significantly higher levels of STRO-1 and CD146, and lower levels of CD105 (Figure 1A). Additionally, SHED showed significantly high numbers of single colony clusters (colony-forming units-fibroblastic; CFU-F) and an elevated cell proliferation rate compared to BMMSCs (Figures 1D and 1E). This elevated proliferative capacity may be associated with the significantly increased telomerase activity in SHED (Figure 1F).

23To compare osteogenic differentiation of SHED with BMMSCs, multiple colony-derived SHED at passage 3 were supplemented with L-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate to induce mineralization in vitro as described previously. After one week of induction, SHED were similar to BMMSCs, showing significantly increased alkaline phosphatase (ALP) activity (Figure 2A) and the number of ALP-positive cells by flow cytometric analysis (Figure 2B), and expression of elevated levels of ALP, Runt related transcription factor 2 (Runx2), dentin sialoprotein (DSP), and osteocalcin (OCN) by immunoblot analysis (Figure 2C). Alizarin Red-positive nodule formation in SHED and BMSMC cultures was notified after four weeks of osteogenic induction, indicating calcium accumulation in vitro (Figures 2D and 2E). However, SHED suffered remarkable impairment of adipogenic differentiation, as shown by decreased numbers of lipid-specific Oil red O-positive cells and reduced expression of adipocyte-specific molecules, peroxisome proliferator-activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) when compared to BMMSCs (Figures 2F-H). To validate the capacity of forming mineralized tissue in vivo by SHED, ex vivo expanded-SHED were transplanted into immunocompromised mice with hydroxyapatite/tricalcium phosphate (HA/TCP) as a carrier. SHED formed a similar amount of mineralized tissue and a reduced amount of hematopoietic marrow components when compared to BMMSC transplants (Figures 2I-K). Next, we confirmed that SHED were similar to BMMSCs in activation of multiple signaling pathways, including TGFβ, ERK, Akt, Wnt, and PDGF (Figures 2L-P).

24In order to compare the immunomodulatory capacity of SHED with BMMSCs, anti-CD3/CD28 antibodies with TGFβ/IL-6 were added to the co-cultures of SHED or BMMSCs with naïve T cells, which were purified from human PBMNCs, levels of IL17+IFNg- Th17 cells and IL17 were significantly reduced in SHED and BMMSC groups compared to the naïve T cell group (Figure 3A). It appeared that SHED showed a significant inhibiting effect in reducing IL17 levels when compared to BMMSCs (Figure 3B). Our previous report indicated that activated T cells induce apoptosis of BMMSCs through the Fas/FasL pathway. To determine whether activated T cells also directly impinge on SHED, as occurs in BMMSCs, SHED were co-cultured with human PBMNCs activated by anti-CD3 specific antibody treatment. We found that the activated PBMNCs were able to induce part of SHED death in the co-culture system (Figure 3C). When SHED were separated from PBMNCs using a Transwell co-culture system or treated using anti-FasL neutralizing antibody, SHED failed to show the cell death (Figure 3C), suggesting that direct cell-cell contact and the Fas/FasL pathway are required for inducing SHED death by activated splenocytes. Next, we confirmed that SHED express Fas by immunoblot analysis (Figure 3D). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to confirm that the SHED death was due to cell apoptosis (Figure 3E).

25Our previous study showed that systemic infusion of BMMSCs offers appropriate treatment for SLE disorders in human patients and SLE-like MRL/lpr mice. Here we selected SLE-like mice at 16 weeks of age to infuse SHED for treating SLE disorders using BMMSCs as a control (Figure 4A). It is known that autoantibodies play a crucial role in SLE patients. Our previous study showed a remarkable increase in the levels of autoantibodies including anti-double strand DNA (dsDNA) IgG and IgM antibodies, and anti-nuclear antibody (ANA) in the peripheral blood. As seen in BMMSC transplantation, SHED transplantation resulted in a significant reduction in serum levels of anti-dsDNA IgG and IgM, and ANA antibodies (Figures 4B-D).

26Histological analysis with hematoxylin and eosin, trichrome, and periodic acid-Schiff staining revealed that SHED transplantation was similar to BMMSC transplantation in recovery of SLE-associated renal disorders, such as nephritis with glomerular basal membrane disorder and messangial proliferation in MRL/lpr mice (Figure 4E). ELISA data showed that SHED and BMMSC transplantation was able to reduce the urine C3 level and elevate the serum C3 level (Figure 4F). Also, SHED transplantation significantly reduced urine protein levels compared to BMMSC transplantation (Figure 4G). Moreover, SHED and BMMSC transplantation significantly elevated creatinine levels in urine and reduced creatinine levels in serum (Figure 4H). This experimental evidence indicated that SHED transplantation is an effective approach for treating SLE disorders.

27Tregs prevent pathogenic autoimmunity by suppressing proliferation and production of pro-inflammatory cytokines in effector immune cells, such as helper T-lymphocytes. In contrast, Th17 cells that produce IL17 are inflammatory cells responsible for the pathogenesis of autoimmune diseases and bone destruction. Our previous study suggested that BMMSC transplantation affects the immune balance between Tregs and Th17 cells in SLE-like disorders. Here we found that SHED transplantation showed more significant effect in up-regulating the ratio of Treg and Th17 cells in comparison to BMMSC transplantation in MRL/lpr mice (Figures 5A-C). Both SHED and BMMSC transplantations showed no significant changes in the level of IL10 and IL6 in MRL/lpr mice (Figures 5D and 5E); however, SHED transplantation provided a remarkable reduction of TH17 cells and IL17 level in MRL/lpr mice when compared to BMMSC transplantation (Figures 5C and 5F).

28Our previous study suggested that BMMSC transplantation-mediated therapy in SLE-like mice may associate with the reconstructing trabecular bone. Here we found SHED were also capable of reconstructing trabecular bone in MRL/lpr mice (Figure 6A). In contrast to BMMSC/osteoblast lineage, osteoclasts play a significant role in the maintenance of bone homeostasis by the bone resorption function. We compared SHED transplantation with BMMSC transplantation in inhibiting osteoclast activity in MRL/lpr mice and found that both SHED and BMMSC transplantation were able to reduce the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the distal femur epiphysis of MRL/lpr mice (Figure 6B), serum levels of runt-related NF-κB ligand (RANKL), a critical factor for osteoclastogenesis (Figure 6C), and bone resorption marker C-terminal telopeptides of type I collagen (CTX;) as compared to untreated MRL/lpr mice (Figure 6D).

29BMMSCs have been successfully utilized to treat a variety of human diseases, such as bone fracture, severe aplastic anemia, acute GVHD, and SLE. SLE is a common and potentially fatal immune disease in which autoantibodies damage multiple organs, including the kidneys, cardiovascular system, nervous system, joints, and skin. The pathology of SLE involves the destruction of targeted organ tissues and accumulation of auto-reactive lymphocytes and immune complexes. Although intensity and organ involvement vary significantly among SLE patients, abnormalities of T and B lymphocytes are universal. Moreover, SLE provokes multifaceted immune modulation, including both deficiency and hyperactivity of the immune system. An understanding of the underlying pathology is crucial to developing optimal therapies for the restoration of immune homeostasis without compromising the protective immune responses to pathogens. MRL/lpr mice were generated by the insertion of the early transposable element ETn in the Fas gene, which causes a striking reduction in Fas mRNA expression and is associated clinically with marked acceleration of the lupus-like disease. Levels of circulating immune complexes rise enormously from about three months of age in MRL-lpr/lpr but not in MRL mice. In this study, we used MRL/lpr mice as a SLE mouse model to indicate that SHED are an appropriate population of postnatal stem cells for SLE treatment as seen in BMMSC-mediated therapy.

30SHED are derived from a very accessible tissue resource and capable of providing enough cells for potential clinical application via high proliferation rate and expression of telomerase. The reason that SHED transplantation showed optimal therapeutic effect may be associated with the fact that SHED showed superior immunomodulatory effects compared to BMMSCs in terms of recovering Tregs/Th17 ratio and reducing Th17 cell levels in peripheral blood. In addition, SHED transplantation, as seen in BMMSC transplantation, is capable of recovering trabecular bone and inhibiting osteoclast activity, suggesting that SHED transplantation, as seen in BMMSC transplantation, could lead the reconstruction of osteoblastic niche to improve SLE disorders in SLE patients and a SLE-like murine model. Therefore, SHED may be an appropriate stem cell resource for treating immune disorders via improved immunomodulatory properties. Systemic infusion of SHED fails to show a significant promoting Treg level in SLE-like mice as seen in an in vitro co-culture system, which may be associated with a complex in vivo condition that hardly compares to a simple co-culture system. However, SHED infusion resulted in a significantly up-regulated level of the ratio between Tregs and Th17 cells. This is an important index indicating immunomodulatory function of SHED due to the fact that Tregs prevent autoimmunity and Th17 cells promote autoimmunity and inflammation.

31The transition from deciduous teeth to adult permanent teeth is a unique and dynamic process in which the development and eruption of permanent teeth is coordinated with the resorption of deciduous teeth. We found that exfoliated deciduous tooth crowns contain a remnant of living pulp comprised of a normal dental pulp structure, including connective tissue, blood vessels, and odontoblasts. We demonstrated that these remnants of pulp tissues in exfoliated deciduous teeth contain SHED. These studies provide the first evidence that a naturally occurring exfoliated organ contains stem cells with the ability to form multiple phenotypes, and that these stem cells may offer a unique stem cell resource for potential clinical applications. SHED are very easily acquired from exfoliated teeth and can be expanded ex vivo to achieve sufficient numbers of cells for tissue regeneration such as repairing parietal defects.

32SHED possess similar stem cell properties as those seen in BMMSCs, including osteo/odontogenic and adipogenic differentiation in vitro, forming mineralized tissue in vivo, and expression of extensive mesenchymal stem cell markers. Moreover, systemic SHED transplantation is capable of offering similar, if not better, therapeutic effect on SLE murine model, suggesting that easily accessed SHED may be a feasible stem cell source for stem cell therapies.

33ALP: alkaline phosphatase; BMMSC: bone marrow mesenchymal stem cells; BrdU: bromodeoxyuridine; C3: complement 3; CFU: colony forming units-fibroblastic; CTX: C-terminal telopeptides of type I collagen; DSP: dentin sialoprotein; FCM: flow cytometry; HA/TCP: hydroxyapatite tricalcium phosphate; IL: interleukin; LPL: lipoprotein lipase; OCN: osteocalcin; PBMNCs: peripheral blood mononuclear cells; PCR: polymerase chain reaction; PPARγ2: peroxisome proliferator-activated receptor γ2; Runx2: Runt related transcription factor 2; Th17: T helper 17; TRAP: telomeric repeat amplification protocol; Tregs: regulatory T cells; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; SHED: Stem cells from human exfoliated deciduous teeth; SLE: systemic lupus erythematosus; sRANKL: soluble receptor activator for nuclear factor κB ligand.

34The authors declare that they have no competing interests.

35TY and KA collected and assembled data, and worked on data analysis and interpretation. CC collected and assembled data. YL worked on data analysis and interpretation. YS, SG and SW worked on conception and design, and SS worked on conception and design, data analysis and interpretation and wrote the manuscript.