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Stemness Maintenance Properties in Human Oral Stem Cells after Long-Term Passage

View Article: PubMed Central - PubMed

ABSTRACT

Background. Neural crest-derived mesenchymal stem cells (MSCs) from human oral tissues possess immunomodulatory and regenerative properties and are emerging as a potential therapeutic tool to treat diverse diseases, such as multiple sclerosis, myocardial infarction, and connective tissue damages. In addition to cell-surface antigens, dental MSCs express embryonic stem cell markers as neural crest cells originate from the ectoderm layer. In vitro passages may eventually modify these embryonic marker expressions and other stemness properties, including proliferation. In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers during early (passage 2) and late passages (passage 15) in MSCs obtained from human gingiva, periodontal, and dental pulp tissues. Methods. Cell proliferation assay, beta galactosidase staining, immunocytochemistry, and real-time PCR techniques were applied. Results. Cell proliferation assay showed no difference between early and late passages while senescence markers p16 and p21 were considerably increased in late passage. Embryonic stem cell markers including SKIL, MEIS1, and JARID2 were differentially modulated between P2 and P15 cells. Discussion. Our results suggest that the presence of embryonic and proliferation markers even in late passage may potentially endorse the application of dental-derived MSCs in stem cell therapy-based clinical trials.

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Mesengenic differentiation potential. hPDLSCs, hDPSCs, and hGMSCs induced to osteogenic commitment, stained with alizarin red S solution at P2 (a1, b1, and c1) and P15 (a2, b2, and c2) showed no statistical significative differences among two different culture stages. RUNX-2 and ALP expressions confirm light microscopy observations for osteogenic commitment (a3, b3, and c3). Adipogenic induction analyzed by oil red solution staining demonstrates the presence of lipid vacuoles at cytoplasmic level in cells cultured at early (d1, e1, and f1) and late passages (d2, e2, and f2). Both PPARγ and FABP4 showed no statistical differences after 28 days of culture, under differentiation conditions, at P2 and P15 (d3, e3, and f3). Mag.: 10x, bars: 10 μm.
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fig2: Mesengenic differentiation potential. hPDLSCs, hDPSCs, and hGMSCs induced to osteogenic commitment, stained with alizarin red S solution at P2 (a1, b1, and c1) and P15 (a2, b2, and c2) showed no statistical significative differences among two different culture stages. RUNX-2 and ALP expressions confirm light microscopy observations for osteogenic commitment (a3, b3, and c3). Adipogenic induction analyzed by oil red solution staining demonstrates the presence of lipid vacuoles at cytoplasmic level in cells cultured at early (d1, e1, and f1) and late passages (d2, e2, and f2). Both PPARγ and FABP4 showed no statistical differences after 28 days of culture, under differentiation conditions, at P2 and P15 (d3, e3, and f3). Mag.: 10x, bars: 10 μm.

Mentions: To evaluate osteogenic differentiation, cells at P2 and P15 were stained with alizarin red S solution. Calcium precipitates were detected at same levels in both P2 and P15 of all hPDLSCs, hPDSCs, and hGMSCs (Figures 2(a1), 2(a2), 2(b1), 2(b2), 2(c1), and 2(c2)). To confirm that cells maintain differentiation ability at P2 and P15, RUNX-2 and ALP were analyzed by qRT-PCR (Figures 2(a3), 2(b3), and 2(c3), resp.). In addition, to evaluate differentiation to adipogenic lineage, cells were stained with oil red O solution to highlight lipid droplet accumulation at cytoplasmic level (Figures 2(d1), 2(d2), 2(e1), 2(e2), 2(f1), and 2(f2)). PPARγ and FABP4, adipogenic-related markers, were expressed with no significant differences among P2 and P15 cells (Figures 2(d3), 2(e3), and 2(f3)). Both mesengenic differentiations showed no statistically significant differences between groups.


Stemness Maintenance Properties in Human Oral Stem Cells after Long-Term Passage
Mesengenic differentiation potential. hPDLSCs, hDPSCs, and hGMSCs induced to osteogenic commitment, stained with alizarin red S solution at P2 (a1, b1, and c1) and P15 (a2, b2, and c2) showed no statistical significative differences among two different culture stages. RUNX-2 and ALP expressions confirm light microscopy observations for osteogenic commitment (a3, b3, and c3). Adipogenic induction analyzed by oil red solution staining demonstrates the presence of lipid vacuoles at cytoplasmic level in cells cultured at early (d1, e1, and f1) and late passages (d2, e2, and f2). Both PPARγ and FABP4 showed no statistical differences after 28 days of culture, under differentiation conditions, at P2 and P15 (d3, e3, and f3). Mag.: 10x, bars: 10 μm.
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fig2: Mesengenic differentiation potential. hPDLSCs, hDPSCs, and hGMSCs induced to osteogenic commitment, stained with alizarin red S solution at P2 (a1, b1, and c1) and P15 (a2, b2, and c2) showed no statistical significative differences among two different culture stages. RUNX-2 and ALP expressions confirm light microscopy observations for osteogenic commitment (a3, b3, and c3). Adipogenic induction analyzed by oil red solution staining demonstrates the presence of lipid vacuoles at cytoplasmic level in cells cultured at early (d1, e1, and f1) and late passages (d2, e2, and f2). Both PPARγ and FABP4 showed no statistical differences after 28 days of culture, under differentiation conditions, at P2 and P15 (d3, e3, and f3). Mag.: 10x, bars: 10 μm.
Mentions: To evaluate osteogenic differentiation, cells at P2 and P15 were stained with alizarin red S solution. Calcium precipitates were detected at same levels in both P2 and P15 of all hPDLSCs, hPDSCs, and hGMSCs (Figures 2(a1), 2(a2), 2(b1), 2(b2), 2(c1), and 2(c2)). To confirm that cells maintain differentiation ability at P2 and P15, RUNX-2 and ALP were analyzed by qRT-PCR (Figures 2(a3), 2(b3), and 2(c3), resp.). In addition, to evaluate differentiation to adipogenic lineage, cells were stained with oil red O solution to highlight lipid droplet accumulation at cytoplasmic level (Figures 2(d1), 2(d2), 2(e1), 2(e2), 2(f1), and 2(f2)). PPARγ and FABP4, adipogenic-related markers, were expressed with no significant differences among P2 and P15 cells (Figures 2(d3), 2(e3), and 2(f3)). Both mesengenic differentiations showed no statistically significant differences between groups.

View Article: PubMed Central - PubMed

ABSTRACT

Background. Neural crest-derived mesenchymal stem cells (MSCs) from human oral tissues possess immunomodulatory and regenerative properties and are emerging as a potential therapeutic tool to treat diverse diseases, such as multiple sclerosis, myocardial infarction, and connective tissue damages. In addition to cell-surface antigens, dental MSCs express embryonic stem cell markers as neural crest cells originate from the ectoderm layer. In vitro passages may eventually modify these embryonic marker expressions and other stemness properties, including proliferation. In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers during early (passage 2) and late passages (passage 15) in MSCs obtained from human gingiva, periodontal, and dental pulp tissues. Methods. Cell proliferation assay, beta galactosidase staining, immunocytochemistry, and real-time PCR techniques were applied. Results. Cell proliferation assay showed no difference between early and late passages while senescence markers p16 and p21 were considerably increased in late passage. Embryonic stem cell markers including SKIL, MEIS1, and JARID2 were differentially modulated between P2 and P15 cells. Discussion. Our results suggest that the presence of embryonic and proliferation markers even in late passage may potentially endorse the application of dental-derived MSCs in stem cell therapy-based clinical trials.

No MeSH data available.


Related in: MedlinePlus