Journal of Korean Dental Science

Korean Academy of Dental Science (대한치의학회)
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Effects of Age and Gender on the Viability and Stem Cell Markers, mRNA, and Protein Expression of Bone Marrow-Derived Stem Cells Cultured in Growth Media

  • Lee, Hyunjin (Department of Periodontics, College of Medicine, The Catholic University of Korea) ;
  • Lee, Hyuna (Department of Periodontics, College of Medicine, The Catholic University of Korea) ;
  • Na, Chae-Bin (Department of Periodontics, College of Medicine, The Catholic University of Korea) ;
  • Park, Jun-Beom (Department of Periodontics, College of Medicine, The Catholic University of Korea)
  • Received : 2018.07.24
  • Accepted : 2018.12.20
  • Published : 2018.12.30
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Abstract

Purpose: Bone marrow has long been a source of primary cells. This study was performed to evaluate the effects of age and sex on the cellular viability and expression of stem cell markers of mRNA and on the protein expression of bone marrow stem cells (BMSCs) derived from healthy donors. Materials and Methods: Stem cells were isolated from human bone marrow and plated in culture plates. The shape of the BMSCs was observed under inverted microscope. Quantitative cellular viability was evaluated using a Cell-Counting Kit-8 assay. The expression of stem cell surface markers was tested and a series of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot was performed to evaluate the expression in each group. Result: The shapes of the cells at 20s, 30s, and 50s were similar to each other. No significant changes in cellular viability were noted among different age groups or sex groups. The BMSCs expressed CD44, CD73, and CD90 surface markers but did not express CD14 and CD34. There were no noticeable differences in CD surface markers among the different age groups. The expressions of CD surface markers were similar between men and women. No significant differences in the secretion of vascular endothelial growth factors (VEGFs) were noted at Day 3 between different age groups. qRT-PCR regarding the expression showed differences between the age groups. However, Western blot analysis showed a decrease in expression but did not reach statistical significance (P>0.05). Conclusion: This study clearly showed no significant differences in shape, cell viability, expression of stem cell surface markers, or secretion of human VEGF among different age groups. However, western blot analysis showed a tendency of age-related decrease which did not reach statistical significance. Collectively, autologous or allogeneic BMSCs should be meticulously applied to obtain optimal results regarding age and sex.

Keywords

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Fig. 1. (A) Evaluation of bone marrow stem cells (BMSCs)’s shape on Day 3 in growth media (×100). Scale bars=400 μm. (B) Qualitative cellular viability results under a confocal microscope on Day 4. Live images, dead images, merged images, and central images are provided. (C) Cellular viability using CCK-8 assay at Days 1 and 3 among different age groups. *Statistically significant differences were noted when compared with the results of the 30s group on Day 1. (D) Cellular viability using CCK-8 assay at Days 1 and 3 in males and females. *Statistically significant differences were noted when compared with the results of males on Day 1.

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Fig. 2. (A) Expression of stem cell immunophenotype observed with flow cytometry. The bone marrow stem cells (BMSCs) in all groups expressed CD44, CD73, and CD90 surface antigens but did not express CD14 or CD34. (B) Expression of the stem cell markers SSEA-4. (C) Secretion of vascular endothelial growth factors from BMSCs among different age groups. *Statistically significant differences were noted when compared with the results of the 20s group on Day 2. **Statistically significant differences were noted when compared with the results of the 20s group on Day 2. ***Statistically significant differences were noted when compared with the results of 30s group on Day 2. ****Statistically significant differences were noted when compared with the results of the 30s group on Day 2. (D) Secretion of vascular endothelial growth factors in males and females. *Statistically significant differences were noted when compared with the results of males on Day 3. **Statistically significant differences were noted when compared with the results of males on Day 2. ***Statistically significant differences were noted when compared with the results of females on Day 2.

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Fig. 3. (A) Runx2 gene expression of human bone marrow-derived stem cells (hBMSCs) according to age. *Statistically significant differences were noted when compared with the results of the 20s group on Day 2. (B) Runx2 gene expression of hBMSCs according to sex. (C) Collagen I gene expression of hBMSCs according to age. *Statistically significant differences were noted when compared with the results of the 20s group on Day 2. (D) Collagen I gene expression of hBMSCs according to sex.

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Fig. 4. (A) Western blot analysis to detect the protein expressions of Runx2, collagen I and GAPDH. (B) Quantitative analysis was performed to show levels of Runx2 according to age. (C) Runx2 expression according to sex. (D) Quantitative analysis of collagen I expression according to age.

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