J Periodont Res 2014 All rights reserved
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12228
Isolation and characterization of human mesenchymal stem cells from gingival connective tissue
S. H. Jin1, J. E. Lee1, J-H. Yun2, I. Kim3, Y. Ko1, J. B. Park1 1 Department of Periodontics, College of Medicine, The Catholic University of Korea, Seoul, Korea, 2Division of Periodontology, Department of Dentistry, School of Medicine, Inha University, Incheon, Korea and 3Division of Oral and Maxillofacial Surgery, Department of Dentistry, Uijeongbu St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Uijeongbu, Korea
Jin SH, Lee JE, Yun J-H, Kim I, Ko Y, Park JB. Isolation and characterization of human mesenchymal stem cells from gingival connective tissue. J Periodont Res 2014; doi: 10.1111/jre.12228. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs. Material and Methods: Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis. Results: GMSCs showed spindle-shaped, fibroblast-like morphology, colonyforming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence. Conclusions: In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs’ proliferation and cryopreservation.
Jun-Beom Park, DDS, MSD, PhD, Department of Periodontics, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea Tel: +82 2 2258 6290 Fax: +82 2 537 2374 e-mail: [email protected] Key words: gingiva; mesenchymal stromal cells;
multipotent stem cells; stem cells Accepted for publication July 13, 2014
2
Jin et al.
Recently, many researchers have been interested in overcoming the limitation of existing procedures using new approaches such as tissue engineering (1). Tissue engineering has three fundamental factors, including cells, biomaterials and biochemical factors. Among various cells, mesenchymal stem cells (MSCs) have great potential in tissue engineering. The MSCs is one of adult stem cell types and these cells are multipotent stromal cells that can differentiate into osteoblasts, chondrocytes and adipocytes (2). In the human body, they could be regarded as readily available reservoirs of reparative cells capable of mobilizing, proliferating and differentiating to the appropriate cell type in response to certain signals (3). Therefore, for the regeneration of the damaged tooth supporting tissues, an increased amount of research has focused on the multi-lineage differentiation potential of the MSCs (4). With a high growth capacity, multipotential MSCs hold great promise for the application of tissue regeneration methods clinically (5). MSCs have been isolated from various tissues, including bone marrow (6), adipose tissue (7) and muscle (8). Recently, MSCs have been found in various oral tissues, including dental pulp (2), lamina propria of oral mucosa (9), periodontal ligament (10) and gingiva (11). Among them, gingival tissue, as it is routinely discarded after respective periodontal surgery, is ideal for the isolation of the human MSCs. Thus, many researchers have tried to collect gingiva-derived MSCs (GMSCs) in recent years. The MSCs can be isolated and characterized by their properties. The MSCs can adhere to the plastic in standard culture conditions (12). Though the MSCs can expand without adherence, they require very specific culture conditions to do so (13). Secondly, MSCs can be differentiated to osteoblasts, adipocytes and chondroblasts in vitro (14). Thirdly, MSCs have several specific surface antigens. The antibodies against CD44, CD73, CD90 and CD105 react with the undifferentiated MSCs (15). However, the MSCs do not express hematopoietic and endothelial cell markers such as CD11,
CD14, CD31, CD33, CD34, CD45 or CD133 (16). The main purpose of this study was to isolate and characterize the GMSCs. In addition, the secondary purpose was to suggest modifications to the protocols for isolation of GMSCs. Moreover, within the authors’ knowledge, this is the first report on presenting CD146, CD166 and CD271 in gingival tissue as stem cell markers.
Material and methods Isolation and culture of gingival connective tissue-derived mesenchymal stem cells
Attached keratinized gingival tissues were collected from 12 healthy patients (mean age, 57.8 15.3 years; five males and seven females) undergoing clinical crown lengthening procedures in July and August of 2013. This study received approval from the Institutional Review Board, Seoul St. Mary’s Hospital, College of Medicine, the Catholic University of Korea, Seoul, Republic of Korea (KC11SISI0348) and informed consent was obtained from the patients. The obtained tissues were processed by four different isolation methods (Table 1). To explain more detail in the modified method (Table 1, method D), gingival tissues were immediately placed in sterile phosphate-buffered saline (PBS; Welgene, Daegu, Korea) with 100 U/mL penicillin, and 100 lg/mL streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA) at 4°C. The tissues were
de-epithelialized and minced into 1–2 mm2 fragments and digested in 0.2 lm filtered alpha-modified minimal essential medium (a-MEM; Gibco, Grand Island, NY, USA) containing dispase (1 mg/mL; Sigma-Aldrich Co.) and collagenase IV (2 mg/mL; SigmaAldrich Co.) at 37°C for 30 min. After discarding the first digested cell suspension, the tissues were digested in the same solution for 90 min at 37°C. The cell suspension was filtered with a 70 lm cell strainer (Falcon, Franklin Lakes, NJ, USA) and seeded with a-MEM containing 15% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 lg/mL streptomycin (SigmaAldrich Co.), 200 mM L-glutamine (Sigma-Aldrich Co.) and 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich Co.) in a 75 cm2 tissue culture flask (Corning, Tewksbury MA, USA). The cells were incubated in a 37°C humidified incubator with 5% CO2 and 95% air. After 24 h, the non-adherent cells were washed with PBS, and replaced with fresh medium and fed every 3–4 d. Cytoskeletal morphology of gingival connective tissue-derived mesenchymal stem cells
GMSCs were seeded in an eightchamber slide (BD Biosciences, San Jose, CA, USA) at a density of 1 9 104 cells/chamber and incubated overnight. The next day, GMSCs were fixed with 4% paraformaldehyde (Sigma-Aldrich Co.), and permeabilized in 1% Triton-X100 with PBS for 3 min. The GMSCs were stained with
Table 1. Isolation methods for mesenchymal stem cells (MSCs)
A
B
C D
Method
Reference
The collected tissue was incubated overnight with 2 mg/mL dispase at 4°C, and then the minced tissues were applied 4 mg/mL collagenase IV for 2 h The minced tissues were digested in 0.1% collagenase and 0.2% dispase for 15 min, and the first cell fraction containing some epithelial cells was discarded. Then, tissues were further incubated with enzyme solution for 5, 10, and 15 min and all cell fractions were pooled The minced tissues were digested five times at 30 min in 3 mg/mL collagenase and 4 mg/mL dispase, and all cell fractions were pooled Modified method; The minced tissues were digested in 2 mg/mL collagenase and 1 mg/mL dispase for 30 min, and the first cell fraction containing some epithelial cells was discarded. Then, tissues were further incubated with same enzyme solution for 90 min
Zhang et al. (20) Tomar et al. (25)
Park et al. (26)
Stem cells from the gingiva fluorescein isothiocyanate–phalloidin (Sigma-Aldrich Co.) and mounted with VECTASHIELDâ Mounting Medium with DAPI (Vector laboratories, Burlingame, CA, USA). Cells were observed under a fluorescence microscope (Axiovert 200; Zeiss, Oberkochen, Germany). Colony-forming unit fibroblast assay
Cell suspension was filtered through the 70 lm strainer (Falcon). The single cell suspension was plated into two culture dishes (100 mm2; Corning) at densities of 0.5 and 1 cells/mm2 (50 cells, dish 1; 100 cells, dish 2). After 14 d, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich Co.), and stained with 1% crystal violet. Colonies of 50 or more cells were scored as colony-forming unit fibroblasts. Multipotent differentiation of gingival connective tissue-derived mesenchymal stem cells Osteogenic differentiation— The GMSCs were seeded in a 24-well cell culture plate (Corning) at a density of 1 9 104 cells/well and incubated overnight. The next day, the medium was replaced with osteogenic induction medium supplemented with a-MEM containing 15% fetal bovine serum, 100 lM dexamethasone (Sigma-Aldrich Co.), 10 mM b-glycerophosphate (SigmaAldrich Co.), 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich Co.) and 1% antibiotic/antimycotic (Gibco). The medium was replaced with fresh induction medium every 3–4 d. At days 14 and 21, Alizarin Red S staining (Sigma-Aldrich Co.) was performed to detect calcium formation. Adipogenic differentiation— GMSCs were seeded in a 24-well cell culture plate (Corning) at a density of 2 9 104 cells/well and incubated overnight. The next day, the medium was replaced with adipogenic induction medium (STEMPROâ Adipogenesis Differentiation Kit; Gibco). The medium was replaced with fresh induction medium every 3–4 d. After 14 d, Oil Red O staining (Sigma-Aldrich Co.) was performed to detect the oil globules.
Chondrogenic differentiation— Alcian blue staining: Five microliter droplets containing the GMSCs (passage 2) were seeded in the center of a 24-well cell culture plate (Corning) at a density of 1.6 9 107 cells/mL. The droplets were kept under high humidity for 2 h, and chondrogenic induction medium (STEMPROâ Chondrogenesis Differentiation Kit; Gibco) was added to each well. The medium was replaced with fresh induction medium every 3–4 d. After 14 d, alcian blue staining (Sigma-Aldrich Co.) was performed to detect the presence of cartilage-specific proteoglycan core protein. Immunofluorescence of chondrogenic differentiation markers: Centrifuge tube (15 mL; BD Biosciences) containing suspension GMSCs (passage 4; 1 9 106 cells) was centrifuged at 200 g for 5 min at room temperature. The cells were resuspended with chondrogenic induction medium (STEMPROâ Chondrogenesis Differentiation Kit; Gibco) and centrifuged at 200 g for 5 min at room temperature. The med-
3
ium was replaced with fresh induction medium every 3–4 d. After 14 d, chondrogenic pellets were stained with chondrogenic differentiation markers (anticollagen IIa1, anti-aggrecan; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and secondary antibodies such as NorthernLightsTM 493 (R&D systems, Minneapolis, MN, USA). The specimens were observed under a confocal laser scanning microscope (LSM 510 Meta; Zeiss). Flow cytometric analysis
Approximately 3 9 105 GMSCs were incubated with specific PE-, APC-, BV421-, PerCP-cyTM5.5- or fluorescein isothiocyanate-conjugated mouse monoclonal antibodies for human CD44, CD73, CD90, CD105, CD14, CD45, CD34 and CD19 (BD Biosciences). Flow cytometric analysis was performed with a flow cytometer (FACSCanto II; BD biosciences) and the FCS Express 4 program (De Novo Software, Glendale, CA, USA).
A
B
C
D
Fig. 1. Morphology of the cells. (A) The first adherent cells appeared at day 6. The cells showed spindle-shaped, fibroblast-like morphology under a light microscope (original magnification 950). (B) The gingival connective tissue-derived mesenchymal stem cells (GMSCs) showed high proliferation and formation of colony at day 9. (C) The GMSCs showed high proliferation rate at day 12. (D) The F-actin of GMSCs was stained by fluorescein isothiocyanate–phalloidin and observed under an immunofluorescence microscope. Cytoskeletal morphology of GMSCs showed spindle-shaped, fibroblast-like morphology. Scale bar = 100 lm (original magnification 9200).
4
Jin et al.
Immunofluorescence of stem cell markers
GMSCs were seeded in an eightchamber slide (BD Biosciences) at a density of 2 9 104 cells/chamber and incubated overnight. The next day, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich Co.), and then permeabilized in 1% TritonX100 with PBS for 3 min. GMSCs were stained with anti-STRO-1, antiSSEA-4 (R&D systems), and anti-CD146, anti-CD166, anti-CD271 antibodies (Santa Cruz Biotechnology), and secondary antibodies such as NorthernLightsTM 493, and NorthernLightsTM 557 (R&D systems). The specimens were mounted in VECTASHIELDâ Mounting Medium with DAPI (Vector laboratories) and then observed under a fluorescence micro-
scope (Axiovert 200; Zeiss) and confocal laser scanning microscope (LSM 510 Meta; Zeiss). Data analysis
All data in this study were expressed as the mean SD from at least three independent experiments.
Results Cytoskeletal morphology of gingival connective tissue-derived mesenchymal stem cells
The first adherent cells appeared 4–6 d after initiation of the primary culture. Generally, the primary cells reached 80–90% of confluence at days 12–14 (data not shown). Under optical microscopy, the primary cells
showed spindle-shaped, fibroblastlike morphology (Fig. 1A–C). The GMSCs showed spindle-shaped, fibroblast-like morphology with wellstained F-actins and nuclei (Fig. 1D). Colony-forming unit fibroblast assay
All cultures contained a subpopulation of cells with well-stained colonies (Fig. 2A and 2B). The number of colonies in dish 2 was much greater than that of dish 1 (98 vs. 55). The number of colonies was approximately the same as the number of seeded single cells. Multi-lineage differentiation of gingival connective tissue-derived mesenchymal stem cells Osteogenic differentiation— After Aliz-
arin
Red
S
A
B
C
D
E
F
G
H
I
J
staining,
mineralized
Fig. 2. (A) Colony-forming unit fibroblast assay at day 14. The colonies were stained by 1% Crystal violet. Fifty cells from the plated single cell suspension and 55 colonies were observed. (B) One hundred cells from the plated single cell suspension and 98 colonies were observed. The gingival connective tissue-derived mesenchymal stem cells showed colony-forming potency and plastic-adherent characteristics. (C) Control at day 14 after Alizarin Red S staining (original magnification 9100.). (D) Osteogenic differentiation at day 14 (original magnification 9100). (E) Control at day 21 after Alizarin Red S staining (original magnification 950). (F) Osteogenic differentiation at day 21 (original magnification 950). (G) Control at day 14 after Oil Red O staining. Oil droplets were not shown at day 14. (Original magnification 9100.) (H) Adipogenic differentiation at day 14. Many oil droplets were observed at day 14. (Original magnification 9100.) (I) Magnified view of control at day 14. Oil droplets were not shown at day 14. (Original magnification 9400.) (J) Magnified view of adipogenic differentiation at day 14. Oil droplets were observed in the cytoplasm of differentiated cells. (Original magnification 9400.)
Stem cells from the gingiva extracellular deposits were observed at day 14, with more found at day 21 (Fig. 2C–F).
A
5
B
Adipogenic differentiation— After Oil Red O staining, oil globules were well observed in the cytoplasm of differentiated cells at day 14 (Fig. 2G–J). The globules had many lipid-rich vacuoles. Chondrogenic differentiation— In the chondrogenic medium, a cell pellet formed from a dense cell colony at days 1–2 (data not shown) and we stained it with alcian blue at day 14 (Fig. 3). The center of the pellet was too thick to be translucent (Fig. 3B). In staining of chondrogenic markers, the cell pellets showed well stained aggrecan and collagen type II a1 compared with controls at day 14 (Fig. 3C–J). Flow cytometric analysis
The GMSCs expressed CD44, CD73, CD90 and CD105 surface markers, but did not express CD14, CD19, CD34 and CD45 (Fig. 4a). Expression percentages were ≤ 1% in negative surface markers and > 90% in positive surface markers. Immunofluorescence of stem cell markers
Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and mesenchymal marker (vimentin) by GMSCs could be observed by immunofluorescence analysis (Fig. 4B–G).
Discussion This report clearly showed the potential for use of the GMSCs in tissue engineering. In the previous position paper, three minimal criteria was suggested for defining MSCs (17): (i) adherence to plastic in standard culture conditions; (ii) specific surface antigen expression (≥ 95% expression of CD105, CD73 and CD90; ≤ 2% expression of hematopoietic marker); and (iii) multipotent differentiation potential (osteoblasts, adipocytes,
C
D
E
F
G
H
I
J
Fig. 3. (A) Control at day 14 after alcian blue. Cell pellet was not formed from the pellet culture. (Original magnification 9100.) (B) Chondrogenic differentiation at day 14. Chondrogenic cell pellet was successfully formed. (C) Control cell pellet observed after aggrecan staining (optical view) (original magnification 970). (D) Control cell pellet was observed after aggrecan staining. (E) Chondrogenic differentiation observed after aggrecan staining (optical view). (F) Chondrogenic differentiation observed after aggrecan staining. (G) Control cell pellet observed after ColIIa1 staining (optical view). (H) Control cell pellet was observed after ColIIa1 staining. (I) Chondrogenic differentiation observed after ColIIa1 staining (optical view). (J) Chondrogenic differentiation observed after ColIIa1 staining.
chondroblasts). In this study, the cultured cells isolated from gingival connective tissue satisfied minimal criteria. First, GMSCs displayed plastic adherence and colony-forming potency in the colony-forming unit fibroblast assay. Secondly, GMSCs showed osteogenic, chondrogenic and adipogenic differentiation. Thirdly, GMSCs expressed specific surface antigens such as CD105, CD73, CD90 and CD44, but did not express hematopoietic antigens. The presence of CD105, CD73 and CD90 was suggested as one of the essential criteria for MSCs (17). However, there are questions on the significance of this criterion because these three markers can be found even on non-MSCs, fibroblastic cells and other cell populations (18). SSEA-4, an early embryonic glycolipid antigen and common marker for undifferentiated pluripotent human embryonic
stem cells, have been identified in the adult MSC population (19). Other markers such as STRO-1, CD146, CD166 and CD271 have been also identified as MSC-associated markers (20). Thus, additional surface markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and mesenchymal marker (vimentin) were applied for analysis in this report. In our study, four methods were used to isolate GMSCs with three of them reported previously. Dispase was used overnight in the first report of GMSC (21). Dispase, a neutral protease isolated from culture filtrates of Bacillus polymyxa, has proven to be a rapid, effective, but gentle agent for separating intact epithelial sheets in culture from the substratum (22), and dispase can be used to separate epithelial cells from gingival connective tissue. However, this method has some possibility of overdigestion and
6
Jin et al. A
B
C
D
E
F
G
Fig. 4. (A) Expression of stem cell immunophenotype observed with flow cytometry. The gingival connective tissue-derived mesenchymal stem cells expressed CD44, CD73, CD90 and CD105 surface antigens, but did not express CD14, CD19, CD34 and CD45. (B) Expression of stem cell and mesenchymal markers. SSEA-4 expression (original magnification 9100). (C) STRO-1 expression. (D) Vimentin expression. (E) CD146 expression (original magnification 9200). (F) CD166 expression. (G) CD271 expression.
this may influence cell surface marker expression such as CD3, CD4 and CD8 (23). In our study, 24 h incubation with dispase was omitted to minimize toxic effects to the cells (24,25).
A fast method was suggested by using collagenase and dispase at the same time (26). Gingiva was digested in 0.1% collagenase and 0.2% dispase for 15 min, and the first cell
fraction containing some epithelial cells was discarded. Then, tissues were further incubated with enzyme solution for 5, 10 and 15 min and all cell fractions were pooled. There was a possibility of incomplete digestion from a short digestion time with the inconvenience of collecting the cells many times (26). In our study, modifications were done using 1 mg/mL dispase and 2 mg/mL collagenase IV for 1.5 h. Modification was done by discarding the first digested cell suspension to exclude the epithelial cells (27). There are various sources for adult stem cells. MSCs can be isolated extraorally from bone marrow (28), but this causes pain and morbidity for a small harvest (29). Furthermore, the ratio of adherent cell to nucleated cell is much lower in bone marrow than in other solid mesenchymal tissues (28). Harvesting from oral tissues is easier, yields more stem cells and is relatively painless with local anesthesia (30). Dental pulp and periodontal ligament are other possible sources of stem cells, easily accessible (31), but their supply is limited by the number of teeth. MSCs have received increasing attention in tissue engineering but many difficulties lie in their use. Invasive procedures such as bone surgery or liposuction may be required to obtain the cells from the bone marrow or fatty tissues and MSCs from the umbilical cord origin can be only obtained at a certain stage. In contrast, MSCs from gingiva can be isolated from minimally invasive procedures at any time in life. Thus, gingiva can be a promising alternative source of MSCs. In conclusion, we isolated and characterized stem cells from human gingival connective tissue with a modified protocol. GMSCs showed multipotency with high proliferation and characteristics of MSCs. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs’ proliferation and cryopreservation.
Stem cells from the gingiva
Acknowledgements This project was supported by a grant from the ITI Foundation for the Promotion of Implantology, Switzerland. The authors report no conflicts of interest related to this study.
11.
12.
References 1. Izumi Y, Aoki A, Yamada Y et al. Current and future periodontal tissue engineering. Periodontol 2000 2011;56: 166–187. 2. Ballini A, De Frenza G, Cantore S et al. In vitro stem cell cultures from human dental pulp and periodontal ligament: new prospects in dentistry. Int J Immunopathol Pharmacol 2007;20:9–16. 3. Pountos I, Corscadden D, Emery P, Giannoudis PV. Mesenchymal stem cell tissue engineering: techniques for isolation, expansion and application. Injury 2007;38:S23–S33. 4. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- and gene-based therapeutic strategies for periodontal regenerative medicine. J Periodontol 2011;82:1223–1237. 5. Hynes K, Menicanin D, Gronthos S, Bartold PM. Clinical utility of stem cells for periodontal regeneration. Periodontol 2000 2012;59:203–227. 6. Kuznetsov SA, Friedenstein AJ, Robey PG. Factors required for bone marrow stromal fibroblast colony formation in vitro. Br J Haematol 1997;97: 561–570. 7. Rodriguez AM, Elabd C, Amri EZ, Ailhaud G, Dani C. The human adipose tissue is a source of multipotent stem cells. Biochimie 2005;87:125–128. 8. Wada MR, Inagawa-Ogashiwa M, Shimizu S, Yasumoto S, Hashimoto N. Generation of different fates from multipotent muscle stem cells. Development 2002;129:2987–2995. 9. Marynka-Kalmani K, Treves S, Yafee M et al. The lamina propria of adult human oral mucosa harbors a novel stem cell population. Stem Cells 2010;28: 984–995. 10. Nagatomo K, Komaki M, Sekiya I et al. Stem cell properties of human periodon-
13.
14.
15.
16.
17.
18.
19.
20.
21.
tal ligament cells. J Periodontal Res 2006;41:303–310. Wang F, Yu M, Yan X et al. Gingivaderived mesenchymal stem cell-mediated therapeutic approach for bone tissue regeneration. Stem Cells Dev 2011;20:2093–2102. Colter DC, Class R, DiGirolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plasticadherent cells from human bone marrow. Proc Natl Acad Sci USA 2000;97: 3213–3218. Baksh D, Davies JE, Zandstra PW. Adult human bone marrow-derived mesenchymal progenitor cells are capable of adhesion-independent survival and expansion. Exp Hematol 2003;31:723– 732. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147. Bobis S, Jarocha D, Majka M. Mesenchymal stem cells: characteristics and clinical applications. Folia Histochem Cytobiol 2006;44:215–230. Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 2007;25:2739–2749. Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–317. Wada N, Gronthos S, Bartold PM. Immunomodulatory effects of stem cells. Periodontol 2000 2013;63:198–216. Gang EJ, Bosnakovski D, Figueiredo CA, Visser JW, Perlingeiro RC. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 2007;109:1743–1751. Sacchetti B, Funari A, Michienzi S et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324–336. Zhang Q, Shi S, Liu Y et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammationrelated tissue destruction in experimental colitis. J Immunol 2009;183:7787–7798.
7
22. Stenn KS, Link R, Moellmann G, Madri J, Kuklinska E. Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J Invest Dermatol 1989;93: 287–290. 23. Abuzakouk M, Feighery C, O’Farrelly C. Collagenase and Dispase enzymes disrupt lymphocyte surface molecules. J Immunol Methods 1996;194:211–216. 24. Wang F, Wang Z, Sun X, Wang F, Xu X, Zhang X. Safety and efficacy of dispase and plasmin in pharmacologic vitreolysis. Invest Ophthalmol Vis Sci 2004;45:3286–3290. 25. Karlinsky JB, Catanese A, Honeychurch C, Sherter CB, Hoppin FG, Snider GL. In vitro effects of elastase and collagenase on mechanical properties of hamster lungs. Chest 1976;69:275–276. 26. Tomar GB, Srivastava RK, Gupta N et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine. Biochem Biophys Res Commun 2010;393:377–383. 27. Park JC, Kim JM, Jung IH et al. Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations. J Clin Periodontol 2011;38:721–731. 28. Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007;327:449– 462. 29. Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279– 4295. 30. Verdugo F, Simonian K, D’Addona A, Ponton J, Nowzari H. Human bone repair after mandibular symphysis block harvesting: a clinical and tomographic study. J Periodontol 2010;81: 702–709. 31. Liu HC, E LL, Wang DS et al. Reconstruction of alveolar bone defects using bone morphogenetic protein 2 mediated rabbit dental pulp stem cells seeded on nano-hydroxyapatite/collagen/poly (L-lactide). Tissue Eng Part A 2011;17: 2417–2433.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12228
Isolation and characterization of human mesenchymal stem cells from gingival connective tissue
S. H. Jin1, J. E. Lee1, J-H. Yun2, I. Kim3, Y. Ko1, J. B. Park1 1 Department of Periodontics, College of Medicine, The Catholic University of Korea, Seoul, Korea, 2Division of Periodontology, Department of Dentistry, School of Medicine, Inha University, Incheon, Korea and 3Division of Oral and Maxillofacial Surgery, Department of Dentistry, Uijeongbu St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Uijeongbu, Korea
Jin SH, Lee JE, Yun J-H, Kim I, Ko Y, Park JB. Isolation and characterization of human mesenchymal stem cells from gingival connective tissue. J Periodont Res 2014; doi: 10.1111/jre.12228. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs. Material and Methods: Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis. Results: GMSCs showed spindle-shaped, fibroblast-like morphology, colonyforming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence. Conclusions: In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs’ proliferation and cryopreservation.
Jun-Beom Park, DDS, MSD, PhD, Department of Periodontics, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea Tel: +82 2 2258 6290 Fax: +82 2 537 2374 e-mail: [email protected] Key words: gingiva; mesenchymal stromal cells;
multipotent stem cells; stem cells Accepted for publication July 13, 2014
2
Jin et al.
Recently, many researchers have been interested in overcoming the limitation of existing procedures using new approaches such as tissue engineering (1). Tissue engineering has three fundamental factors, including cells, biomaterials and biochemical factors. Among various cells, mesenchymal stem cells (MSCs) have great potential in tissue engineering. The MSCs is one of adult stem cell types and these cells are multipotent stromal cells that can differentiate into osteoblasts, chondrocytes and adipocytes (2). In the human body, they could be regarded as readily available reservoirs of reparative cells capable of mobilizing, proliferating and differentiating to the appropriate cell type in response to certain signals (3). Therefore, for the regeneration of the damaged tooth supporting tissues, an increased amount of research has focused on the multi-lineage differentiation potential of the MSCs (4). With a high growth capacity, multipotential MSCs hold great promise for the application of tissue regeneration methods clinically (5). MSCs have been isolated from various tissues, including bone marrow (6), adipose tissue (7) and muscle (8). Recently, MSCs have been found in various oral tissues, including dental pulp (2), lamina propria of oral mucosa (9), periodontal ligament (10) and gingiva (11). Among them, gingival tissue, as it is routinely discarded after respective periodontal surgery, is ideal for the isolation of the human MSCs. Thus, many researchers have tried to collect gingiva-derived MSCs (GMSCs) in recent years. The MSCs can be isolated and characterized by their properties. The MSCs can adhere to the plastic in standard culture conditions (12). Though the MSCs can expand without adherence, they require very specific culture conditions to do so (13). Secondly, MSCs can be differentiated to osteoblasts, adipocytes and chondroblasts in vitro (14). Thirdly, MSCs have several specific surface antigens. The antibodies against CD44, CD73, CD90 and CD105 react with the undifferentiated MSCs (15). However, the MSCs do not express hematopoietic and endothelial cell markers such as CD11,
CD14, CD31, CD33, CD34, CD45 or CD133 (16). The main purpose of this study was to isolate and characterize the GMSCs. In addition, the secondary purpose was to suggest modifications to the protocols for isolation of GMSCs. Moreover, within the authors’ knowledge, this is the first report on presenting CD146, CD166 and CD271 in gingival tissue as stem cell markers.
Material and methods Isolation and culture of gingival connective tissue-derived mesenchymal stem cells
Attached keratinized gingival tissues were collected from 12 healthy patients (mean age, 57.8 15.3 years; five males and seven females) undergoing clinical crown lengthening procedures in July and August of 2013. This study received approval from the Institutional Review Board, Seoul St. Mary’s Hospital, College of Medicine, the Catholic University of Korea, Seoul, Republic of Korea (KC11SISI0348) and informed consent was obtained from the patients. The obtained tissues were processed by four different isolation methods (Table 1). To explain more detail in the modified method (Table 1, method D), gingival tissues were immediately placed in sterile phosphate-buffered saline (PBS; Welgene, Daegu, Korea) with 100 U/mL penicillin, and 100 lg/mL streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA) at 4°C. The tissues were
de-epithelialized and minced into 1–2 mm2 fragments and digested in 0.2 lm filtered alpha-modified minimal essential medium (a-MEM; Gibco, Grand Island, NY, USA) containing dispase (1 mg/mL; Sigma-Aldrich Co.) and collagenase IV (2 mg/mL; SigmaAldrich Co.) at 37°C for 30 min. After discarding the first digested cell suspension, the tissues were digested in the same solution for 90 min at 37°C. The cell suspension was filtered with a 70 lm cell strainer (Falcon, Franklin Lakes, NJ, USA) and seeded with a-MEM containing 15% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 lg/mL streptomycin (SigmaAldrich Co.), 200 mM L-glutamine (Sigma-Aldrich Co.) and 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich Co.) in a 75 cm2 tissue culture flask (Corning, Tewksbury MA, USA). The cells were incubated in a 37°C humidified incubator with 5% CO2 and 95% air. After 24 h, the non-adherent cells were washed with PBS, and replaced with fresh medium and fed every 3–4 d. Cytoskeletal morphology of gingival connective tissue-derived mesenchymal stem cells
GMSCs were seeded in an eightchamber slide (BD Biosciences, San Jose, CA, USA) at a density of 1 9 104 cells/chamber and incubated overnight. The next day, GMSCs were fixed with 4% paraformaldehyde (Sigma-Aldrich Co.), and permeabilized in 1% Triton-X100 with PBS for 3 min. The GMSCs were stained with
Table 1. Isolation methods for mesenchymal stem cells (MSCs)
A
B
C D
Method
Reference
The collected tissue was incubated overnight with 2 mg/mL dispase at 4°C, and then the minced tissues were applied 4 mg/mL collagenase IV for 2 h The minced tissues were digested in 0.1% collagenase and 0.2% dispase for 15 min, and the first cell fraction containing some epithelial cells was discarded. Then, tissues were further incubated with enzyme solution for 5, 10, and 15 min and all cell fractions were pooled The minced tissues were digested five times at 30 min in 3 mg/mL collagenase and 4 mg/mL dispase, and all cell fractions were pooled Modified method; The minced tissues were digested in 2 mg/mL collagenase and 1 mg/mL dispase for 30 min, and the first cell fraction containing some epithelial cells was discarded. Then, tissues were further incubated with same enzyme solution for 90 min
Zhang et al. (20) Tomar et al. (25)
Park et al. (26)
Stem cells from the gingiva fluorescein isothiocyanate–phalloidin (Sigma-Aldrich Co.) and mounted with VECTASHIELDâ Mounting Medium with DAPI (Vector laboratories, Burlingame, CA, USA). Cells were observed under a fluorescence microscope (Axiovert 200; Zeiss, Oberkochen, Germany). Colony-forming unit fibroblast assay
Cell suspension was filtered through the 70 lm strainer (Falcon). The single cell suspension was plated into two culture dishes (100 mm2; Corning) at densities of 0.5 and 1 cells/mm2 (50 cells, dish 1; 100 cells, dish 2). After 14 d, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich Co.), and stained with 1% crystal violet. Colonies of 50 or more cells were scored as colony-forming unit fibroblasts. Multipotent differentiation of gingival connective tissue-derived mesenchymal stem cells Osteogenic differentiation— The GMSCs were seeded in a 24-well cell culture plate (Corning) at a density of 1 9 104 cells/well and incubated overnight. The next day, the medium was replaced with osteogenic induction medium supplemented with a-MEM containing 15% fetal bovine serum, 100 lM dexamethasone (Sigma-Aldrich Co.), 10 mM b-glycerophosphate (SigmaAldrich Co.), 10 mM ascorbic acid 2-phosphate (Sigma-Aldrich Co.) and 1% antibiotic/antimycotic (Gibco). The medium was replaced with fresh induction medium every 3–4 d. At days 14 and 21, Alizarin Red S staining (Sigma-Aldrich Co.) was performed to detect calcium formation. Adipogenic differentiation— GMSCs were seeded in a 24-well cell culture plate (Corning) at a density of 2 9 104 cells/well and incubated overnight. The next day, the medium was replaced with adipogenic induction medium (STEMPROâ Adipogenesis Differentiation Kit; Gibco). The medium was replaced with fresh induction medium every 3–4 d. After 14 d, Oil Red O staining (Sigma-Aldrich Co.) was performed to detect the oil globules.
Chondrogenic differentiation— Alcian blue staining: Five microliter droplets containing the GMSCs (passage 2) were seeded in the center of a 24-well cell culture plate (Corning) at a density of 1.6 9 107 cells/mL. The droplets were kept under high humidity for 2 h, and chondrogenic induction medium (STEMPROâ Chondrogenesis Differentiation Kit; Gibco) was added to each well. The medium was replaced with fresh induction medium every 3–4 d. After 14 d, alcian blue staining (Sigma-Aldrich Co.) was performed to detect the presence of cartilage-specific proteoglycan core protein. Immunofluorescence of chondrogenic differentiation markers: Centrifuge tube (15 mL; BD Biosciences) containing suspension GMSCs (passage 4; 1 9 106 cells) was centrifuged at 200 g for 5 min at room temperature. The cells were resuspended with chondrogenic induction medium (STEMPROâ Chondrogenesis Differentiation Kit; Gibco) and centrifuged at 200 g for 5 min at room temperature. The med-
3
ium was replaced with fresh induction medium every 3–4 d. After 14 d, chondrogenic pellets were stained with chondrogenic differentiation markers (anticollagen IIa1, anti-aggrecan; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and secondary antibodies such as NorthernLightsTM 493 (R&D systems, Minneapolis, MN, USA). The specimens were observed under a confocal laser scanning microscope (LSM 510 Meta; Zeiss). Flow cytometric analysis
Approximately 3 9 105 GMSCs were incubated with specific PE-, APC-, BV421-, PerCP-cyTM5.5- or fluorescein isothiocyanate-conjugated mouse monoclonal antibodies for human CD44, CD73, CD90, CD105, CD14, CD45, CD34 and CD19 (BD Biosciences). Flow cytometric analysis was performed with a flow cytometer (FACSCanto II; BD biosciences) and the FCS Express 4 program (De Novo Software, Glendale, CA, USA).
A
B
C
D
Fig. 1. Morphology of the cells. (A) The first adherent cells appeared at day 6. The cells showed spindle-shaped, fibroblast-like morphology under a light microscope (original magnification 950). (B) The gingival connective tissue-derived mesenchymal stem cells (GMSCs) showed high proliferation and formation of colony at day 9. (C) The GMSCs showed high proliferation rate at day 12. (D) The F-actin of GMSCs was stained by fluorescein isothiocyanate–phalloidin and observed under an immunofluorescence microscope. Cytoskeletal morphology of GMSCs showed spindle-shaped, fibroblast-like morphology. Scale bar = 100 lm (original magnification 9200).
4
Jin et al.
Immunofluorescence of stem cell markers
GMSCs were seeded in an eightchamber slide (BD Biosciences) at a density of 2 9 104 cells/chamber and incubated overnight. The next day, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich Co.), and then permeabilized in 1% TritonX100 with PBS for 3 min. GMSCs were stained with anti-STRO-1, antiSSEA-4 (R&D systems), and anti-CD146, anti-CD166, anti-CD271 antibodies (Santa Cruz Biotechnology), and secondary antibodies such as NorthernLightsTM 493, and NorthernLightsTM 557 (R&D systems). The specimens were mounted in VECTASHIELDâ Mounting Medium with DAPI (Vector laboratories) and then observed under a fluorescence micro-
scope (Axiovert 200; Zeiss) and confocal laser scanning microscope (LSM 510 Meta; Zeiss). Data analysis
All data in this study were expressed as the mean SD from at least three independent experiments.
Results Cytoskeletal morphology of gingival connective tissue-derived mesenchymal stem cells
The first adherent cells appeared 4–6 d after initiation of the primary culture. Generally, the primary cells reached 80–90% of confluence at days 12–14 (data not shown). Under optical microscopy, the primary cells
showed spindle-shaped, fibroblastlike morphology (Fig. 1A–C). The GMSCs showed spindle-shaped, fibroblast-like morphology with wellstained F-actins and nuclei (Fig. 1D). Colony-forming unit fibroblast assay
All cultures contained a subpopulation of cells with well-stained colonies (Fig. 2A and 2B). The number of colonies in dish 2 was much greater than that of dish 1 (98 vs. 55). The number of colonies was approximately the same as the number of seeded single cells. Multi-lineage differentiation of gingival connective tissue-derived mesenchymal stem cells Osteogenic differentiation— After Aliz-
arin
Red
S
A
B
C
D
E
F
G
H
I
J
staining,
mineralized
Fig. 2. (A) Colony-forming unit fibroblast assay at day 14. The colonies were stained by 1% Crystal violet. Fifty cells from the plated single cell suspension and 55 colonies were observed. (B) One hundred cells from the plated single cell suspension and 98 colonies were observed. The gingival connective tissue-derived mesenchymal stem cells showed colony-forming potency and plastic-adherent characteristics. (C) Control at day 14 after Alizarin Red S staining (original magnification 9100.). (D) Osteogenic differentiation at day 14 (original magnification 9100). (E) Control at day 21 after Alizarin Red S staining (original magnification 950). (F) Osteogenic differentiation at day 21 (original magnification 950). (G) Control at day 14 after Oil Red O staining. Oil droplets were not shown at day 14. (Original magnification 9100.) (H) Adipogenic differentiation at day 14. Many oil droplets were observed at day 14. (Original magnification 9100.) (I) Magnified view of control at day 14. Oil droplets were not shown at day 14. (Original magnification 9400.) (J) Magnified view of adipogenic differentiation at day 14. Oil droplets were observed in the cytoplasm of differentiated cells. (Original magnification 9400.)
Stem cells from the gingiva extracellular deposits were observed at day 14, with more found at day 21 (Fig. 2C–F).
A
5
B
Adipogenic differentiation— After Oil Red O staining, oil globules were well observed in the cytoplasm of differentiated cells at day 14 (Fig. 2G–J). The globules had many lipid-rich vacuoles. Chondrogenic differentiation— In the chondrogenic medium, a cell pellet formed from a dense cell colony at days 1–2 (data not shown) and we stained it with alcian blue at day 14 (Fig. 3). The center of the pellet was too thick to be translucent (Fig. 3B). In staining of chondrogenic markers, the cell pellets showed well stained aggrecan and collagen type II a1 compared with controls at day 14 (Fig. 3C–J). Flow cytometric analysis
The GMSCs expressed CD44, CD73, CD90 and CD105 surface markers, but did not express CD14, CD19, CD34 and CD45 (Fig. 4a). Expression percentages were ≤ 1% in negative surface markers and > 90% in positive surface markers. Immunofluorescence of stem cell markers
Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and mesenchymal marker (vimentin) by GMSCs could be observed by immunofluorescence analysis (Fig. 4B–G).
Discussion This report clearly showed the potential for use of the GMSCs in tissue engineering. In the previous position paper, three minimal criteria was suggested for defining MSCs (17): (i) adherence to plastic in standard culture conditions; (ii) specific surface antigen expression (≥ 95% expression of CD105, CD73 and CD90; ≤ 2% expression of hematopoietic marker); and (iii) multipotent differentiation potential (osteoblasts, adipocytes,
C
D
E
F
G
H
I
J
Fig. 3. (A) Control at day 14 after alcian blue. Cell pellet was not formed from the pellet culture. (Original magnification 9100.) (B) Chondrogenic differentiation at day 14. Chondrogenic cell pellet was successfully formed. (C) Control cell pellet observed after aggrecan staining (optical view) (original magnification 970). (D) Control cell pellet was observed after aggrecan staining. (E) Chondrogenic differentiation observed after aggrecan staining (optical view). (F) Chondrogenic differentiation observed after aggrecan staining. (G) Control cell pellet observed after ColIIa1 staining (optical view). (H) Control cell pellet was observed after ColIIa1 staining. (I) Chondrogenic differentiation observed after ColIIa1 staining (optical view). (J) Chondrogenic differentiation observed after ColIIa1 staining.
chondroblasts). In this study, the cultured cells isolated from gingival connective tissue satisfied minimal criteria. First, GMSCs displayed plastic adherence and colony-forming potency in the colony-forming unit fibroblast assay. Secondly, GMSCs showed osteogenic, chondrogenic and adipogenic differentiation. Thirdly, GMSCs expressed specific surface antigens such as CD105, CD73, CD90 and CD44, but did not express hematopoietic antigens. The presence of CD105, CD73 and CD90 was suggested as one of the essential criteria for MSCs (17). However, there are questions on the significance of this criterion because these three markers can be found even on non-MSCs, fibroblastic cells and other cell populations (18). SSEA-4, an early embryonic glycolipid antigen and common marker for undifferentiated pluripotent human embryonic
stem cells, have been identified in the adult MSC population (19). Other markers such as STRO-1, CD146, CD166 and CD271 have been also identified as MSC-associated markers (20). Thus, additional surface markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and mesenchymal marker (vimentin) were applied for analysis in this report. In our study, four methods were used to isolate GMSCs with three of them reported previously. Dispase was used overnight in the first report of GMSC (21). Dispase, a neutral protease isolated from culture filtrates of Bacillus polymyxa, has proven to be a rapid, effective, but gentle agent for separating intact epithelial sheets in culture from the substratum (22), and dispase can be used to separate epithelial cells from gingival connective tissue. However, this method has some possibility of overdigestion and
6
Jin et al. A
B
C
D
E
F
G
Fig. 4. (A) Expression of stem cell immunophenotype observed with flow cytometry. The gingival connective tissue-derived mesenchymal stem cells expressed CD44, CD73, CD90 and CD105 surface antigens, but did not express CD14, CD19, CD34 and CD45. (B) Expression of stem cell and mesenchymal markers. SSEA-4 expression (original magnification 9100). (C) STRO-1 expression. (D) Vimentin expression. (E) CD146 expression (original magnification 9200). (F) CD166 expression. (G) CD271 expression.
this may influence cell surface marker expression such as CD3, CD4 and CD8 (23). In our study, 24 h incubation with dispase was omitted to minimize toxic effects to the cells (24,25).
A fast method was suggested by using collagenase and dispase at the same time (26). Gingiva was digested in 0.1% collagenase and 0.2% dispase for 15 min, and the first cell
fraction containing some epithelial cells was discarded. Then, tissues were further incubated with enzyme solution for 5, 10 and 15 min and all cell fractions were pooled. There was a possibility of incomplete digestion from a short digestion time with the inconvenience of collecting the cells many times (26). In our study, modifications were done using 1 mg/mL dispase and 2 mg/mL collagenase IV for 1.5 h. Modification was done by discarding the first digested cell suspension to exclude the epithelial cells (27). There are various sources for adult stem cells. MSCs can be isolated extraorally from bone marrow (28), but this causes pain and morbidity for a small harvest (29). Furthermore, the ratio of adherent cell to nucleated cell is much lower in bone marrow than in other solid mesenchymal tissues (28). Harvesting from oral tissues is easier, yields more stem cells and is relatively painless with local anesthesia (30). Dental pulp and periodontal ligament are other possible sources of stem cells, easily accessible (31), but their supply is limited by the number of teeth. MSCs have received increasing attention in tissue engineering but many difficulties lie in their use. Invasive procedures such as bone surgery or liposuction may be required to obtain the cells from the bone marrow or fatty tissues and MSCs from the umbilical cord origin can be only obtained at a certain stage. In contrast, MSCs from gingiva can be isolated from minimally invasive procedures at any time in life. Thus, gingiva can be a promising alternative source of MSCs. In conclusion, we isolated and characterized stem cells from human gingival connective tissue with a modified protocol. GMSCs showed multipotency with high proliferation and characteristics of MSCs. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs’ proliferation and cryopreservation.
Stem cells from the gingiva
Acknowledgements This project was supported by a grant from the ITI Foundation for the Promotion of Implantology, Switzerland. The authors report no conflicts of interest related to this study.
11.
12.
References 1. Izumi Y, Aoki A, Yamada Y et al. Current and future periodontal tissue engineering. Periodontol 2000 2011;56: 166–187. 2. Ballini A, De Frenza G, Cantore S et al. In vitro stem cell cultures from human dental pulp and periodontal ligament: new prospects in dentistry. Int J Immunopathol Pharmacol 2007;20:9–16. 3. Pountos I, Corscadden D, Emery P, Giannoudis PV. Mesenchymal stem cell tissue engineering: techniques for isolation, expansion and application. Injury 2007;38:S23–S33. 4. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- and gene-based therapeutic strategies for periodontal regenerative medicine. J Periodontol 2011;82:1223–1237. 5. Hynes K, Menicanin D, Gronthos S, Bartold PM. Clinical utility of stem cells for periodontal regeneration. Periodontol 2000 2012;59:203–227. 6. Kuznetsov SA, Friedenstein AJ, Robey PG. Factors required for bone marrow stromal fibroblast colony formation in vitro. Br J Haematol 1997;97: 561–570. 7. Rodriguez AM, Elabd C, Amri EZ, Ailhaud G, Dani C. The human adipose tissue is a source of multipotent stem cells. Biochimie 2005;87:125–128. 8. Wada MR, Inagawa-Ogashiwa M, Shimizu S, Yasumoto S, Hashimoto N. Generation of different fates from multipotent muscle stem cells. Development 2002;129:2987–2995. 9. Marynka-Kalmani K, Treves S, Yafee M et al. The lamina propria of adult human oral mucosa harbors a novel stem cell population. Stem Cells 2010;28: 984–995. 10. Nagatomo K, Komaki M, Sekiya I et al. Stem cell properties of human periodon-
13.
14.
15.
16.
17.
18.
19.
20.
21.
tal ligament cells. J Periodontal Res 2006;41:303–310. Wang F, Yu M, Yan X et al. Gingivaderived mesenchymal stem cell-mediated therapeutic approach for bone tissue regeneration. Stem Cells Dev 2011;20:2093–2102. Colter DC, Class R, DiGirolamo CM, Prockop DJ. Rapid expansion of recycling stem cells in cultures of plasticadherent cells from human bone marrow. Proc Natl Acad Sci USA 2000;97: 3213–3218. Baksh D, Davies JE, Zandstra PW. Adult human bone marrow-derived mesenchymal progenitor cells are capable of adhesion-independent survival and expansion. Exp Hematol 2003;31:723– 732. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147. Bobis S, Jarocha D, Majka M. Mesenchymal stem cells: characteristics and clinical applications. Folia Histochem Cytobiol 2006;44:215–230. Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 2007;25:2739–2749. Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–317. Wada N, Gronthos S, Bartold PM. Immunomodulatory effects of stem cells. Periodontol 2000 2013;63:198–216. Gang EJ, Bosnakovski D, Figueiredo CA, Visser JW, Perlingeiro RC. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 2007;109:1743–1751. Sacchetti B, Funari A, Michienzi S et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324–336. Zhang Q, Shi S, Liu Y et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammationrelated tissue destruction in experimental colitis. J Immunol 2009;183:7787–7798.
7
22. Stenn KS, Link R, Moellmann G, Madri J, Kuklinska E. Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J Invest Dermatol 1989;93: 287–290. 23. Abuzakouk M, Feighery C, O’Farrelly C. Collagenase and Dispase enzymes disrupt lymphocyte surface molecules. J Immunol Methods 1996;194:211–216. 24. Wang F, Wang Z, Sun X, Wang F, Xu X, Zhang X. Safety and efficacy of dispase and plasmin in pharmacologic vitreolysis. Invest Ophthalmol Vis Sci 2004;45:3286–3290. 25. Karlinsky JB, Catanese A, Honeychurch C, Sherter CB, Hoppin FG, Snider GL. In vitro effects of elastase and collagenase on mechanical properties of hamster lungs. Chest 1976;69:275–276. 26. Tomar GB, Srivastava RK, Gupta N et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine. Biochem Biophys Res Commun 2010;393:377–383. 27. Park JC, Kim JM, Jung IH et al. Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations. J Clin Periodontol 2011;38:721–731. 28. Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007;327:449– 462. 29. Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13:4279– 4295. 30. Verdugo F, Simonian K, D’Addona A, Ponton J, Nowzari H. Human bone repair after mandibular symphysis block harvesting: a clinical and tomographic study. J Periodontol 2010;81: 702–709. 31. Liu HC, E LL, Wang DS et al. Reconstruction of alveolar bone defects using bone morphogenetic protein 2 mediated rabbit dental pulp stem cells seeded on nano-hydroxyapatite/collagen/poly (L-lactide). Tissue Eng Part A 2011;17: 2417–2433.
