Comparison of Cellular and Differentiation Characteristics of Mesenchymal Stem Cells Derived from Human Gingiva and Periodontal Ligament

Abstract

Objectives: Dental tissues possess multipotent stem cells with varying biological properties. The present study was aimed to establish a primary culture of human gingiva-derived mesenchymal stem cells (GMSCs) and periodontal ligament-derived stem cells (PDLSCs) from periodontally healthy subjects and compare their biological characteristics.

Materials and methods: Gingival and periodontal ligament (PDL) tissues were collected from extracted premolar teeth of five healthy subjects and primary cultures were established. Basic biological characteristics, such as cell morphology, viability, proliferation capacity, and colony-forming units, and in vitro osteogenic and adipogenic differentiation potential were performed at passage 3 of GMSCs and PDLSCs. This was followed by immuno-phenotyping and flow cytometric analysis for identification of positive mesenchymal stem cell (MSC) markers, such as CD73, CD90, and CD105, and negative markers CD45 and CD34.

Statistical analysis used: One-way analysis of variance (ANOVA).

Results: Primary cultures of GMSCs and PDLSCs were successfully established. Cells exhibited a fibroblast-like morphology with a homogeneous population at passage 3. Cells derived from both tissues were highly viable (>95%), proliferative, and capable of forming colonies. Both cells did not exhibit any noticeable differences in cellular properties. Immunofluorescence and flow cytometric analyses showed positivity for MSC markers, CD73, CD90, and CD105, and negativity for CD34 and CD45. Furthermore, GMSCs and PDLSCs were capable of differentiating in vitro into osteocytes as evidenced by Alizarin red-S staining, and adipocytes as demonstrated by oil red O staining.

Conclusions: The results of the present study indicate that both GMSCs and PDLSCs have similar cellular characteristics and mesenchymal differentiation potential. Therefore, they may serve as an equally potent source of stem cells for use in cell-based periodontal therapies.

Keywords: Comparative characterization; gingiva; periodontal ligament; stem cells.

Figure 1

Establishment of primary culture and morphological features of GMSCs and PDLSCs. (A and C) After the initiation of primary culture, tissue fragments adhered onto the plastic culture dish and the migration of round and spindle-shaped cells was observed after 24–48 h. (B) During the next five days of culture, cells released from tissue explants exhibited fibroblast-like morphology. (D) After two weeks of primary culture, cells reached near confluence status (×100)

Figure 2

Viability assay and colony forming ability of GMSCs and PDLSCs. (A) Viability assay was performed by trypan-blue exclusion method from passage 1–5 using a hemocytometer. Values are represented as means ± standard deviation (SD) of triplicates at each passage. Cells from all the passages of GMSCs and PDLSCs exhibited >97% viability and no significant difference (P>0.05) was observed between passages as well as cell lines. (B). Macroscopic image of colony-forming ability of GMSCs at 15 days of culture. Cells were stained with Crystal violet

Figure 3

Proliferation assay and population doubling time (PDT) of GMSCs and PDLSCs. (A) Cell proliferation assay was performed by counting the cells at Days 0, 3, 6, 9, and 12 using a hemocytometer. Values are represented as means ± standard deviation (SD) of triplicates at each time point. (B) PDT was calculated using a standard formula. Differences in PDT between the cell lines were analyzed by Sidak’s multiple comparison test. P<0.05, indicates significant difference

Figure 4

Expression of stem cell markers in GMSCs (A–F) and PDLSCs (G–L) by immunofluorescence analysis. Membrane localization of cell surface antigens reveals immunoreactivity for (A and G) CD73 (FITC, positive), (B and H) CD90 (FITC, Thy-1, positive), and (C and I) CD105 (Alexa Fluor, endoglin, weakly positive). Both cells were negative for (D and J) CD34 (FITC, negative) and (E and K) CD45 (FITC, negative). (F and L) Representative images showing the staining of nucleus by propidium iodide (PI) in GMSCs and PDLSCs, respectively (×20 and ×40)

Figure 5

Flow cytometry analysis of stem cell marker expression in GMSCs (A) and PDLSCs (B). Cells were stained with antibody against CD73, CD90, CD105, CD34, and CD45. In merged images, dark-lined histograms indicate signal of isotype control, and green and orange-lined histograms indicate the positive reactivity with stained specific antibody. A total of 10,000 cells were analyzed for each sample in duplicates. Representative examples indicating marker expression profiles are presented

Figure 6

Osteogenic and adipogenic differentiation potential of GMSCs and PDLSCs. (A and C) GMSCs and PDLSCs without osteogenic induction medium (control). (B and D) Images indicating the calcium deposition and mineralization of nodules by osteocytes (arrows) differentiated from GMSCs and PDLSCs after 3 weeks induction and demonstrated by Alizarin red-S staining (arrows) (×20). (E and G) Cells with no adipogenic induction medium. (F and H) Formation of intracellular lipid droplets in adipocytes was observed when GMSCs and PDLSCS were cultured in adipogenic induction media for 3 weeks. Fat globules presence was confirmed by oil red-O staining (arrows) (×20)