Immunomodulatory properties of stem cells from human exfoliated deciduous teeth
- Takayoshi Yamaza†1, 2,
- Akiyama Kentaro†1,
- Chider Chen1,
- Yi Liu1,
- Yufang Shi3,
- Stan Gronthos4,
- Songlin Wang5 and
- Songtao Shi1Email author
© Yamaza et al.; licensee BioMed Central Ltd. 2010
Received: 18 July 2009
Accepted: 15 March 2010
Published: 15 March 2010
Stem cells from human exfoliated deciduous teeth (SHED) have been identified as a population of postnatal stem cells capable of differentiating into osteogenic and odontogenic cells, adipogenic cells, and neural cells. Herein we have characterized mesenchymal stem cell properties of SHED in comparison to human bone marrow mesenchymal stem cells (BMMSCs).
We used in vitro stem cell analysis approaches, including flow cytometry, inductive differentiation, telomerase activity, and Western blot analysis to assess multipotent differentiation of SHED and in vivo implantation to assess tissue regeneration of SHED. In addition, we utilized systemic SHED transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice.
We found that SHED are capable of differentiating into osteogenic and adipogenic cells, expressing mesenchymal surface molecules (STRO-1, CD146, SSEA4, CD73, CD105, and CD166), and activating multiple signaling pathways, including TGFβ, ERK, Akt, Wnt, and PDGF. Recently, BMMSCs were shown to possess an immunomodulatory function that leads to successful therapies for immune diseases. We examined the immunomodulatory properties of SHED in comparison to BMMSCs and found that SHED had significant effects on inhibiting T helper 17 (Th17) cells in vitro. Moreover, we found that SHED transplantation is capable of effectively reversing SLE-associated disorders in MRL/lpr mice. At the cellular level, SHED transplantation elevated the ratio of regulatory T cells (Tregs) via Th17 cells.
These data suggest that SHED are an accessible and feasible mesenchymal stem cell source for treating immune disorders like SLE.
Human 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 [1–5]. 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 [6–11]. 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) [12–14], ameliorating Hematopoietic Stem Cell engraftment [15, 16], and systemic lupus erythematosus (SLE) . Recently, mesenchymal stem cells derived from other tissues have also been found to possess immunomodulatory functions [18–20] which offer opportunities to find more effective and feasible mesenchymal stem cell sources for cell therapies.
Stem 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.
Materials and methods
C57BL/6J and C3MRL-Fas lpr /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).
Human tooth, bone marrow and peripheral blood samples
Human 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).
Isolation and culture of SHED and BMMSCs
Mononuclear cells isolated from the remnant dental pulp tissue of the deciduous incisors were cultured as reported previously [21, 24]. BMMSCs culture was described previously . The detailed protocols were described in Additional file 1.
Cell surface markers analysis
The 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.
Colony forming units-fibroblastic (CFU-F) assay
Cell proliferation assay
Telomerase activity assay
In vitro osteogenic induction assay
Osteogenic differentiation assays of SHED and BMMSCs were performed according to previous publications [21, 28]. Osteogenic markers and mineralized nodule formation were assessed as described previously [[21, 28] and Additional file 1].
Adipogenic induction assay in vitro
In vivo osteogenic differentiation
Xenogeneic transplantation was performed using immunocompromised mice as described [21, 25, 26]. 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.
Co-culture of human PBMNCs or T lymphocytes with SHED or BMMSCs
PBMNCs 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
Xenogeneic SHED or human BMMSCs into MRL/lpr mice
Under 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).
FCM analysis of Treg and Th17 cells
Measurement of biomarkers in culture supernatant, blood serum and urine
Several 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].
Histological analysis of kidney and bone
Kidneys 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].
All 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.
Antibodies and primers
SHED possess mesenchymal stem cell properties
Interplays between SHED and T-lymphocytes
SHED transplantation improves SLE phenotypes in MRL/lpr mice
Histological 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.
SHED transplantation regulates ratio of Tregs and Th17 cells
BMMSCs 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 [35–37]. 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.
SHED 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 .
The 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 .
SHED 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.
bone marrow mesenchymal stem cells
colony forming units-fibroblastic
C-terminal telopeptides of type I collagen
hydroxyapatite tricalcium phosphate
peripheral blood mononuclear cells
polymerase chain reaction
peroxisome proliferator-activated receptor γ2
Runt related transcription factor 2
T helper 17
telomeric repeat amplification protocol
regulatory T cells
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling
Stem cells from human exfoliated deciduous teeth
systemic lupus erythematosus
soluble receptor activator for nuclear factor κB ligand.
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 (R01DE017449 to S.S. and ARRA R01DE019413 to S.S. and Y.S.).
- Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997, 276: 71-74. 10.1126/science.276.5309.71.View ArticlePubMed
- Bianco P, Riminucci M, Gronthos S, Robey PG:Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells. 2001, 19: 180-192. 10.1634/stemcells.19-3-180.View ArticlePubMed
- Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV: Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation. 1974, 17: 331-340. 10.1097/00007890-197404000-00001.View ArticlePubMed
- Owen M, Friedenstein AJ: Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp. 1988, 136: 42-60.PubMed
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284: 143-147. 10.1126/science.284.5411.143.View ArticlePubMed
- Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, Hardy W, Devine S, Ucker D, Deans R, Moseley A, Hoffman R: Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 2002, 30: 42-48. 10.1016/S0301-472X(01)00769-X.View ArticlePubMed
- Uccelli A, Pistoia V, Moretta L: Mesenchymal stem cells: A new strategy for immunosuppression?. Trends Immunol. 2007, 28: 219-226. 10.1016/j.it.2007.03.001.View ArticlePubMed
- Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, Cazzanti F, Risso M, Gualandi F, Mancardi GL, Pistoia V, Uccelli A: Human mesenchymal stem cells modulate B-cell functions. Blood. 2006, 107: 367-372. 10.1182/blood-2005-07-2657.View ArticlePubMed
- Rasmusson I, Le Blanc K, Sundberg B, Ringdén O: Mesenchymal stem cells stimulate antibody secretion in human B cells. Scand J Immunol. 2007, 65: 336-343. 10.1111/j.1365-3083.2007.01905.x.View ArticlePubMed
- Ramasamy R, Fazekasova H, Lam EW, Soeiro I, Lombardi G, Dazzi F: Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle. Transplantation. 2007, 83: 71-76. 10.1097/01.tp.0000244572.24780.54.View ArticlePubMed
- Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L: Mesenchymalstemcell-natural killer cell interactions: Evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood. 2006, 107: 1484-1490. 10.1182/blood-2005-07-2775.View ArticlePubMed
- Aggarwal S, Pittenger MF: Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005, 105: 1815-1822. 10.1182/blood-2004-04-1559.View ArticlePubMed
- Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringdén O: Developmental Committee of the European Group for Blood and Marrow Transplantation: Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet. 2004, 363: 1439-1441. 10.1016/S0140-6736(04)16104-7.View ArticlePubMed
- Chen X, Armstrong MA, Li G: Mesenchymal stem cells in immunoregulation. Immunol Cell Biol. 2006, 84: 413-421. 10.1111/j.1440-1711.2006.01458.x.View ArticlePubMed
- Koç ON, Gerson SL, Cooper BW, Laughlin M, Meyerson H, Kutteh L, Fox RM, Szekely EM, Tainer N, Lazarus HM: Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol. 2000, 18: 307-316.PubMed
- Noort WA, Kruisselbrink AB, in't Anker PS, Kruger M, van Bezooijen RL, de Paus RA, Heemskerk MH, Löwik CW, Falkenburg JH, Willemze R, Fibbe WE: Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34 cells in NOD/SCID mice. Exp Hematol. 2002, 30: 870-878. 10.1016/S0301-472X(02)00820-2.View ArticlePubMed
- Sun L, Akiyama K, Zhang H, Yamaza T, Hou Y, Zhao S, Xu T, Le A, Shi S: Mesenchymal Stem Cell Transplantation Reverses Multi-Organ Dysfunction in Systemic Lupus Erythematosus Mice and Humans. Stem Cells. 2009, 27: 1421-1432. 10.1002/stem.68.PubMed CentralView ArticlePubMed
- Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, Kawamata M, Kato T, Okochi H, Ochiya T: IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells. 2008, 26: 2705-2712. 10.1634/stemcells.2008-0034.View ArticlePubMed
- Cho KS, Park HK, Park HY, Jung JS, Jeon SG, Kim YK, Roh HJ: IFATS collection: Immunomodulatory effects of adipose tissue-derived stem cells in an allergic rhinitis mouse model. Stem Cells. 2009, 27: 259-265. 10.1634/stemcells.2008-0283.View ArticlePubMed
- Wada N, Menicanin D, Shi S, Bartold PM, Gronthos S: Immunomodulatory properties of human periodontal ligament stem cells. J Cell Physiol. 2009, 219: 667-676. 10.1002/jcp.21710.View ArticlePubMed
- Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S: SHED: Stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA. 2003, 100: 5807-5812. 10.1073/pnas.0937635100.PubMed CentralView ArticlePubMed
- Laino G, Graziano A, d'Aquino R, Pirozzi G, Lanza V, Valiante S, De Rosa A, Naro F, Vivarelli E, Papaccio G: An approachable human adult stem cell source for hard-tissue engineering. J Cell Physiol. 2006, 206: 693-701. 10.1002/jcp.20526.View ArticlePubMed
- Zheng Y, Liu Y, Zhang CM, Zhang HY, Li WH, Shi S, Le AD, Wang SL: Stem cells from deciduous tooth repair mandibular defect in swine. J Dent Res. 2009, 88: 249-254. 10.1177/0022034509333804.PubMed CentralView ArticlePubMed
- Seo BM, Sonoyama W, Coppe C, Kikuiri T, Akiyama K, Lee JS, Shi S: SHED repair critical-size calvarial defects in immunocompromised mice. Oral Diseases. 2008, 14: 428-434. 10.1111/j.1601-0825.2007.01396.x.PubMed CentralView ArticlePubMed
- Yamaza T, Miura Y, Akiyama K, Bi Y, Sonoyama W, Gronthos S, Chen W, Le A, Shi S: Mesenchymal Stem Cell-Mediated Ectopic Hematopoiesis Alleviates Aging-Related Phenotype in Immunocompromised Mice. Blood. 2009, 113: 2595-2604. 10.1182/blood-2008-10-182246.PubMed CentralView ArticlePubMed
- Shi S, Gronthos S, Chen S, Reddi A, Counter CM, Robey PG, Wang CY: Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol. 2002, 20: 587-591. 10.1038/nbt0602-587.View ArticlePubMed
- Gronthos S, Mankani M, Brahim J, Robey PG, Shi S: Post-natal dental pulp stem cells in vivo and in vitro. Proc Natl Acad Sci USA. 2000, 97: 13625-13630. 10.1073/pnas.240309797.PubMed CentralView ArticlePubMed
- Yamaza T, Miura Y, Bi Y, Liu Y, Akiyama K, Sonoyama W, Patel V, Gutkind S, Young M, Gronthos S, Le A, Wang CY, Chen W, Shi S: Pharmacologic stem cell based intervention as a new approach to osteoporosis treatment in rodents. PLoS ONE. 2008, 3: e2615-10.1371/journal.pone.0002615.PubMed CentralView ArticlePubMed
- Liu Y, Zheng Y, Ding G, Fang D, Zhang C, Bartold PM, Gronthos S, Shi S, Wang S: Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem Cells. 2008, 26: 1065-1073. 10.1634/stemcells.2007-0734.PubMed CentralView ArticlePubMed
- La Cava A: T-regulatory cells in systemic lupus erythematosus. Lupus. 2008, 17: 421-425. 10.1177/0961203308090028.View ArticlePubMed
- Garrett-Sinha LA, John S, Gaffen SL: IL-17 and the Th17 lineage in systemic lupus erythematosus. Curr Opin Rheumatol. 2008, 20: 519-525. 10.1097/BOR.0b013e328304b6b5.View ArticlePubMed
- Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y, Kadono Y, Tanaka S, Kodama T, Akira S, Iwakura Y, Cua DJ, Takayanagi H: Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J Exp Med. 2006, 203: 2673-2682. 10.1084/jem.20061775.PubMed CentralView ArticlePubMed
- El-Badri NS, Hakki A, Ferrari A, Shamekh R, Good RA: Autoimmune disease: is it a disorder of the microenvironment?. Immunol Res. 2008, 41: 79-86. 10.1007/s12026-007-0053-8.View ArticlePubMed
- Cordeiro AC, Isenberg DA: Novel therapies in lupus - focus on nephritis. Acta Reumatol Port. 2008, 33: 157-169.PubMed
- Rahman A, Isenberg DA: Systemic Lupus Erythematosus. N Engl J Med. 2008, 358: 929-939. 10.1056/NEJMra071297.View ArticlePubMed
- Kyttaris VC, Juang YT, Tsokos GC: Immune cells and cytokines in systemic lupus erythematosus: an update. Curr Opin Rheumatol. 2005, 17: 518-522. 10.1097/01.bor.0000170479.01451.ab.View ArticlePubMed
- Crispin JC, Tsokos GC: Novel molecular targets in the treatment of systemic lupus erythematosus. Autoimmun Rev. 2008, 7: 256-261. 10.1016/j.autrev.2007.11.020.PubMed CentralView ArticlePubMed
- Ramanujam M, Davidson A: Targeting of the immune system in systemic lupus erythematosus. Expert Rev Mol Med. 2008, 10: e2-10.1017/S1462399408000562.View ArticlePubMed
- Neubert K, Meister S, Moser K, Weisel F, Maseda D, Amann K, Wiethe C, Winkler TH, Kalden JR, Manz RA, Voll RE: The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis. Nat Med. 2008, 14: 748-755. 10.1038/nm1763.View ArticlePubMed
- Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, Cheroutre H: Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science. 2007, 317: 256-260. 10.1126/science.1145697.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.