|Year : 2021 | Volume
| Issue : 2 | Page : 175-180
Comparison of stem cell markers on Gingival mesenchymal stem cells among diabetic and healthy individuals. An Ex Vivo Pilot study
Suman Basavaraju, Shashank Chandanala, Dhakshaini Mysuru Rajashekar
Department of Periodontology, J. S. S. Dental College & Hospital, J. S. S. Academy of Higher Education and Research, Mysuru, Karnataka, India
|Date of Submission||04-Aug-2020|
|Date of Decision||18-Aug-2020|
|Date of Acceptance||05-Dec-2020|
|Date of Web Publication||17-Apr-2021|
Dr. Suman Basavaraju
Department of Periodontology, J. S. S. Dental College & Hospital, J. S. S. Academy of Higher Education and Research, Mysuru 570015, Karnataka.
Source of Support: None, Conflict of Interest: None
Aim: Diabetes mellitus (DM), being a risk factor for periodontal disease, affects various cellular functions, and it plays an important role in periodontal regeneration. However, the effect of hyperglycemia on Gingival mesenchymal stem cells (GMSCs) is unclear. We aim at investigating the effect of hyperglycemia on stem cell markers of GMSCs compared with normoglycemic conditions. Materials and Methods: An ex vivo pilot study was conducted by obtaining primary gingival tissues from subjects. Subjects from the outpatient department of periodontology were randomly selected according to the criteria. Three subjects with an HbA1c of >6.5 were selected as the test group, and three subjects with an HbA1c <6 served as controls. The tissue was enzymatically digested in 0.5 mg/mL collagenase (Sigma-Aldrich) and cultured in Knockout Dulbecco‘s Modified Eagles Medium (DMEM - KO medium, Life Technologies). MSC-like cells isolated were examined under a microscope and investigated for specific cell surface antigens CD73, CD90, CD105, CD34, CD45, HLA ABC, and HLA DR by using flow cytometry. Data were analyzed by a two-tailed non-parametric Mann–Whitney U test using SPS software. P < 0.05 was considered statistically significant. Results: The immunophenotype characterization in both test and control exhibited positive expression of CD73 (94.95%,95.93%), CD90 (98.05%, 88.53%), CD105 (73.97%, 73.88%), and HLA ABC (98.83%,98.16%) and negative expression of CD34 (0.02%,0.10%), CD45(0.57%,1.72%), and HLA DR (0.64%, 2.57%). Statistical analysis by the nonparametric Mann–Whitney U test did not reveal any statistically significant difference in the expression. Conclusion: GMSCs from the hyperglycemic environment retained their stem cell characteristics by positive expression of CD73, CD90, CD105, and HLA ABC, and negative expression of CD34, CD45, and HLA DR.
Keywords: Diabetes, Gingival Mesenchymal Stem Cells (GMSCs), Periodontal Healing, Stem Cell Markers, Stem Cells
|How to cite this article:|
Basavaraju S, Chandanala S, Rajashekar DM. Comparison of stem cell markers on Gingival mesenchymal stem cells among diabetic and healthy individuals. An Ex Vivo Pilot study. J Int Oral Health 2021;13:175-80
|How to cite this URL:|
Basavaraju S, Chandanala S, Rajashekar DM. Comparison of stem cell markers on Gingival mesenchymal stem cells among diabetic and healthy individuals. An Ex Vivo Pilot study. J Int Oral Health [serial online] 2021 [cited 2021 Jun 17];13:175-80. Available from: https://www.jioh.org/text.asp?2021/13/2/175/313843
| Introduction|| |
Gingiva is a unique oral tissue attached to the alveolar bone and is recognized as a biological mucosal barrier with distinctive immunomodulatory properties. Healing in gingiva is characterized by marked reduced inflammation, rapid re-epithelialization, and fetal-like scarless healing as reported by Larjava et al. Studies by Pitaru et al. and Haekkinen et al. have demonstrated the capacity of adult gingival wound fibroblasts to produce extracellular matrix proteins in response to growth factors implied by the existence of a resident heterogeneous cell population. Later, it was Zhang et al. who isolated progenitor cells in the gingiva and named them GMSCs. The cells demonstrated biological properties that fulfilled the minimal criteria for human MSCs as proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposed by Dominici et al. The GMSCs have been the most sought mesenchymal stem cells (MSCs) for tissue engineering due to their noninvasive procurement and easy availability.
Studies have shown that Bone Marrow Mesenchymal Stem Cells (BM-MSCs) have demonstrated reduced proliferation potential in response to pathophysiological mechanisms, but the effects of these alterations on GMSCs are unclear,,,,,; hence, it is imperative to study the effect of these unfavorable environments on GMSCs.
Being a metabolic syndrome, DM is characterized by hyperglycemia, resulting in nephropathy, neuropathy, retinopathy, vasculopathy, delayed wound healing, and periodontitis. Hyperglycemia is known to affect the periodontal tissues through direct effects and indirectly by formation of advanced glycation end-products (AGEs), leading to an overall impairment of wound healing. Hyperglycemia has also been reported to reduce proliferation, migration, and differentiation potential of periodontal ligament cells (PDLCs), gingival fibroblasts (GFs), and MSCs. However, the effects of hyperglycemia on GMSCs in diabetic individuals need to be elucidated.
Hence, this pilot study aimed at assessing the effects of hyperglycemia on the stemness of GMSCs in diabetics by assessing its stem cell marker expression by flow cytometry.
| Materials and Methods|| |
Recruitment of subjects
Patients visiting the outpatient department of Periodontology were screened. Subjects in the age group of 18 to 50 years advised for extraction/gingivectomy/crown lengthening were selected for the study. Within the scope of the study, only diabetic status was considered and no importance was given to the periodontal status of the subjects. Subjects with an HbA1c of >6.5 were selected as the test group, and five subjects with an HbA1c<6 served as controls. Patients with a diabetic history of a minimum of two years were included. Prediabetic subjects and diabetics with other systemic complications were excluded from the study. However, as the samples were processed, two samples were excluded as they were contaminated during the assay procedure.
Processing of samples
After obtaining consent from patients, gingival tissue was surgically excised and transported to the laboratory. Tissue was washed in phosphate-buffered saline (PBS) solution, cut into small pieces (~2 mm × 2 mm), and digested by incubating with 0.5 mg/mL collagenase (Sigma-Aldrich) for 3 hours at 37°C. The digested tissue was washed with PBS several times and plated in DMEM - KO medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Himedia), Glutamax (Life Technologies), antibiotics (100 U mL−1 penicillin, 100 µg mL−1 streptomycin), and 1% amphotericin. The flasks were incubated in 5% carbon dioxide at 37°C, and the cells were allowed to grow. The culture flasks were periodically checked by using phase-contrast inverted microscopy, and the basic medium was changed three times per week.
Immunophenotype characterization of gingival cells by flow cytometry
The immunophenotype was determined by flow cytometry. A total of 0.5 × 106 gingival cells at passage three or four were incubated with specific individual monoclonal antibodies; they were conjugated with fluorescein (FITC), phycoerythrin (PE), or peridinin chlorophyll protein in 250µl PBS for 30min in the dark at room temperature. The primary antibodies used were cluster of differentiation (CD) 90, CD73, CD45, CD34, CD105, HLA DR, and HLA ABC (BD pharmingen). Cells were then diluted in 4µl PBS, centrifuged, and suspended with 600µl PBS–formaldehyde 2%. Acquisition and analysis were performed with a flow cytometer by using cell quest pro software. Isotype controls used were immunoglobulin G (IgG)1 fluorescein isothiocyanate and IgG1 PE monoclonal antibodies (BD pharmingen).
All data were expressed as mean ± SD and analyzed by a two-tailed nonparametric Mann–Whitney U test using SPS software. P <0.05 was considered statistically significant [Table 1].
|Table 1: Flow cytometry analysis of surface markers expressed by test and control|
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| Results|| |
Microscopic appearance of cultured cells
Under optic microscopy, the initial culture showed small, rounded cells and spindle-shaped cells. After 48 h, spindle-shaped fibroblast-like cells that were tightly adhered and well spread were seen in both the diabetic and control groups. Colonies were seen at around day 5 and became 70 and 80% confluent between 12 and 15 days. From the first passage, cultivated cells homogeneously showed a fibroblast-like spindle shape [Figure 1].
|Figure 1: Phase contrast image of GMSCs of control and test shows presence of homogeneous fibroblast like population. A and B are representative experimental groups|
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The immunophenotype characterization was evaluated from the culture at the third through fifth passages. In each of the three paired cultivated samples, positive immunostaining was consistently obtained for gingival MSCs, including CD90, CD105, CD73, and HLA ABC, and negative immunostaining or weakly positive staining was obtained for typical hematopoietic markers, including CD45, CD34, and HLA-DR [Figure 2].
|Figure 2: Comparitive flow cytometry analysis of cell surface markers of GMSCs for control and test. Both the groups showed positive expression of CD73, CD90, CD105, HLA ABC and negative for CD34, CD45, HLA DR in three different experimental groups (A-C)|
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Both test and control exhibit positive expression for CD73 (94.95%,95.93%), CD90 (98.05%, 88.53%), CD105 (73.97%, 73.88%), and HLA ABC (98.83%, 98.16%) and negative expression for CD34 (0 .02%, 0.10%), CD45 (0.57%, 1.72%), and HLA DR (0.64%, 2.57%) [Table 1].
| Discussion|| |
MSCs are nonhematopoietic stromal cells that can differentiate into and contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. MSCs exhibit properties that make these cells potentially ideal candidates for tissue engineering. The adult human MSCs can express CD105 (SH2), CD73 (SH3/4), CD44, CD90 (Thy-1), CD71, and Stro-1 as well as the adhesion molecules CD106 (VCAM-1), CD166 (ALCAM), ICAM-1, and CD29 but they do not express the hematopoietic markers CD45, CD34, CD14 or CD11 or CD80, CD86, CD40 (co-stimulatory molecules), or CD31 (PECAM-1), CD18 (Leu-CAM), CD56 (NCAM-1) (adhesion molecules).,,,
Pathological conditions induced by dysregulation result in aberrant functions of stem cells. Studies have reported the effects of hyperglycemia on MSCs., GMSCs are also exposed to a diabetic environment in the oral cavity. However there is no research on the effects of hyperglycemia on the characteristics of GMSCs in vivo. In our study, we aimed at detecting the expression of surface markers of GMSCs in diabetic patients to assess whether hyperglycemia did have any effect on the stemness of GMSCs.
In our study, GMSCs isolated from diabetic and healthy individuals showed positive expression of CD90, CD73, CD105, and HLA ABC and negative expression of hematopoietic markers CD34, CD45, and HLA DR, which is in accordance with studies highlighting the stem cell marker profile of GMSCs.,Stemcell marker profile was immunopositive for CD29 (>91.2 ± 1.73%), CD44 (>86.4 ± 5.76%), CD73 (>86.8 ± 2.9%), CD90 (>82 ± 9.59%), and CD105 (~37.6 ± 15.56%) for diabetic BMMSCs and it was comparable with that of healthy samples.
Statistical analysis in our study did not show any significant difference in the stem cell marker expression between diabetic and healthy GMSCs. This is in comparison to the study that reported no difference in the expression of stemcell markers between diabetic and healthy dermal cells. One study compared Type 1 diabetic BM-MSCs with their healthy counterparts and reported that T1D-MSCs showed similar morphology, immunophenotypic characteristics, adipocyte differentiation potential, expression of immunomodulatory genes, and in vitro immunosuppressive capacity.
Our study expressed good percentages of CD73, CD90, CD105, and HLA ABC in both the groups. In his pioneering study, Pittenger was the first to propose definitive markers for MSCs, with the initial ones being CD73, CD105 along with CD90. These are the primary molecules used to identify MSCs by the ISCT. The advantage of these primary molecules is that cultured MSCs are uniformly and strongly positive for CD105, CD90, and CD73, regardless of their passage or time in culture or with little difference between donors. CD73 (ecto-5-nucleotidase) is one of the classic markers that specifically defines the MSC population with the capacity to metabolize extracellular nucleotides and a pivotal regulator of the local immune response.MSCs derived from murine pericardial adipose tissue (pMSC) constituted CD73 high and CD73 low pMSC populations. CD73 high pMSC brought about structural and functional repair when implanted in the myocardial infarcted rat and displayed a pronounced anti-inflammatory activity by attenuating CCR2+ macrophage infiltration and upregulating several anti-inflammatory genes. The high expression of CD73 in diabetic GMSCs in our study could be suggestive of the cells retaining their reparative and anti-inflammatory property. CD90, also known as Thy-1, a cell-anchored glycoprotein, has a role in cell–cell–matrix interactions and cell motility. A high percentage of expression of CD90 in spermatogonial stem cells (SSCs, 85.9%) and hair follicle stem cells (HFSCs, 50.85%) confirmed their role in stem cell growth and differentiation. However, one study reports high expression of CD90 in MSCs, stating that CD90 may play an important role in maintaining the undifferentiated state of MSCs and confirmed that a reduction in CD90 expression leads to a more efficient osteogenic differentiation, irrespective of the source. We could speculate that the comparative reduction in percentage expression of CD90 in the control group in our study, though not statistically significant, may suggest a better osteogenic differentiation potential in comparison with the test group. Another MSC marker CD105 (endoglin), also known as SH2, is involved in cell proliferation, differentiation, and migration and is a component of the receptor complex of transforming growth factor-beta (TGF-β)., MSCs with CD105+ have shown osteogenic, adipogenic, and chondrogenic differentiation potential. Our data showed positive expression of this marker in both the healthy and diabetic samples. Contrary to our study, reduced expression of CD105 in diabetic adipose stem cells (ADSC) was noted and has suggested the reduced angiogenic potency of diabetic ADSCs. In one study, diabetes altered the adipose stem cell (ASC) niche in situ and diabetic murine ASC were compromised in their ability for vasculogenesis. They identified a subpopulation of cells that was diminished in both type 1 and type 2 models of diabetes that were characterized by the high expression of genes known to be important for new blood vessel growth. The immunological characterization was assessed by the expression of major histocompatibility complex I and II, HLA ABC, and HLA DR, respectively. Although expression of HLA-ABC did not vary in the GMSCs of patients with diabetes, there was a significant reduction in the expression of HLA-DR, which might comprimise their ability to suppress inflammation prevalent during periodontitis. Thus, it could be possible that hyperglycemia-induced reduction in HLA-DR on GMSCs alters their ability to suppress the persistent inflammation observed in periodontitis, thus hindering repair and regeneration of gingival tissue. Our study showed more than 95% expression of HLA ABC and negative expression (<2%) of HLA DR. Our study demonstrated that hyperglycemia did not alter the immunosuppressive property of GMSCs. Evidence that the human embryonic stem cell-derived MSCs shared similar immunogenicity and immunosuppressive abilities with BMSCs, but differed in the expression profile of immunological markers and the responsiveness to certain inflammatory cytokines has been reported. Our study throws light on stem cell marker expression of GMSCs from a diabetic environment that is comparable to a normoglycemic environment. Hyperglycemia did not alter the stem cell marker expression of GMSCs.
As a pilot study, a small sample was recruited, which can be further expanded to obtain more statistically significant results. Within the scope of this study,we could speculate that diabetic cells retained their stemness. However, further research can be carried out to identify their differentiative potencies, thereby obtaining therapeutic benefits in the field of regenerative medicine.
| Conclusion|| |
As per the results of the study, we can conclude that hyperglycemia will not alter the stem cell marker expression of GMSCs. The GMSCs from patients with diabetes exhibited stem cell marker expression comparable to that of normoglycemic GMSCs.
Currently, GMSCs has no explicit surface marker constellation for MSCs’ characterization. Though for standardization purposes, studies commonly refer to the marker arrangement proposed by the ISCT, additional marker lists have been added. The identification of specific stem cell markers for GMSCs could provide more authentic results for GMSCs’ identification. G-MSCs marker expression alteration under different settings/ culturing conditions poses a challenge. Further research is needed for the development of culture techniques and settings that could positively influence their cellular properties before transplantation. Our current understanding stems from an in vitro cell culture that does not entirely translate to human clinical situations. A further deeper understanding of the biological processes and interactions of GMSCs and a hyperglycemic environment is required before GMSCs-based regenerative therapies could translate to clinical settings. The results of the current ex vivo study can accelerate further research, which can help translate these findings for clinical use.
The authors extend their heartfelt gratitude to Dr. Akhila Prashanth, Professor and Mr. Anshu MSc, Research Scholar, Department of Biochemistry, J.S.S. Medical College and Hospital for their constant support. They are indebted to Dr. Jyothi S. Prasanna, Additional Professor, Manipal Institute of Regenerative Medicine for her guidance and encouragement.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
SBV: Content Design, Definition of intellectual content, Literature search, Experimental Studies, Data acquisition, and Article preparation. SC: Experimental Studies, Data analysis, Statistical analysis, and Article preparation. DMR: Def of intellectual content, Literarture search, Clinical studies, and Article preparation. All the authors had reviewed and given approval for publication.
Ethical policy and institutional review board statement
Ethical clearance was obtained from the Institutional Ethical Committee (JSSDCH/Ethical/26/2016–17. Date: 10-11-2016).
Patient declaration of consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/ have given his/ her/ their consent for his/ her/ their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Data availability statement
Data will be available on request from the author ([email protected]).
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[Figure 1], [Figure 2]