Hydrogen sulphide increases hepatic differentiation of human tooth pulp stem cells compared with human bone marrow stem cells.

AIM: To determine the differences in stem cell properties, in hepatic differentiation and in the effects of hydrogen sulphide (H2 S) on hepatic differentiation between human bone marrow stem cells (hBMC) and stem cells from human exfoliated primary tooth pulp (SHED). METHODOLOGY: CD117(+) cells were magnetically separated and subjected to hepatic differentiation. CD117(+) cell lineages were characterized for transcription factors indicative of stem cells by qRT-PCR. For the last 9 days of the differentiation, the test cells were exposed to 0.1 ng mL(-1) H2 S. Immunocytochemistry and flow cytometry of albumin, alpha-fetoprotein and carbamoyl phosphate synthetase were carried out after differentiation. Urea concentration and glycogen synthesis were also determined. RESULTS: Genes expressed in SHED were also expressed in BMC. No difference in expression level of hepatic markers was shown by immunofluorescence. SHED showed more positive cells than hBMC (P < 0.01). H2 S increased the number of positive cells in both cultures (P < 0.01). Urea concentration and glycogen synthesis increased significantly after H2 S exposure (P < 0.001 and P < 0.05, respectively). Real-time PCR data were analysed by RT(2) profiler RT-PCR Array Data Analysis version 3.5 (Qiagen), and ELISA data were analysed by Bonferroni's multiple comparison using Windows spss version 16 (SPSS Inc, Chicago, IL, USA). Bonferroni's multiple comparison test was also carried out after angle transformation for the percentage data of flow cytometer using Windows spss(®) version 16 (SPSS Inc). Statistical significance was accepted at P < 0.05. CONCLUSIONS: Stem cells from human exfoliated primary tooth pulp and BMC have similar properties. The level of hepatic differentiation in SHED compared with BMC was the same or higher. H2 S increased the level of hepatic differentiation.

p53-Pathway activity and apoptosis in hydrogen sulfide-exposed stem cells separated from human gingival epithelium.

BACKGROUND AND OBJECTIVE: Hydrogen sulfide ( H2S ) is a volatile sulfur compound responsible for physiological halitosis. H2S was also reported as having periodontal pathologic activities. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; oral keratinocyte stem cells OKSCs play key roles in maintaining this barrier. The p53 pathway is responsible for regulating key biological events. Increased apoptosis and cell-cycle arrest of DNA repair can affect keratinocyte stem cells, having a direct impact on the architecture of the oral epithelial tissue. However, the link between H2S , p53 activity and OKSCs has not yet been fully explored. The main objective of the present study was to explore the implications of the p53 pathway in OKSCs following exposure to H2S. MATERIAL AND METHODS: OKSCs were isolated from human gingival epithelium and incubated with physiological levels of H2S for 24 and 48 h. Apoptosis and the mitochondrial membrane potential were detected using flow cytometry. Cytochrome c, total p53, phosphorylated p53 and caspase activity were assessed using specific ELISAs. p53 Pathway gene activity was assayed using quantitative RT-PCR. RESULTS: The levels of apoptosis were significantly increased following incubation in the presence of H2S, especially after 48 h (36.95 ± 1.91% vs. 4.77 ± 0.74%). Caspases 9 and 3 were activated, whereas caspase-8 activity remained low. Total p53 activity and particularly phosphorylated p53 at serine 46, were significantly enhanced compared with controls (47.11 ± 9.84 units/mL vs. 1.5 ± 0 units/mL and 32.22 ± 10.23 units/mL vs. 0.15 ± 0 units/mL, respectively, at 48 h). Among p53 pathway genes, apoptosis-related genes [i.e. phosphatase and tensin homolog ( PTEN ), B-cell CLL/lymphoma 2 ( BCL2), sirtuin 3 ( SIRT3) and caspases]) were dramatically increased when compared with controls. Moreover, cell-cycle progression genes [i.e. E2F transcription factor (E2F) family and histone deacetylase ( HDAC )] and DNA-repair genes [i.e. growth arrest and DNA-damage-inducible, gamma ( GADD45G ) family and serine/threonine-protein kinase Chk2 ( CHEK2)] were also increased. CONCLUSION: Following incubation with H2 S , OKSCs express multiple p53-associated genes, including programmed cell death, cell-cycle control and DNA-repair genes.

Characterization of oral keratinocyte stem cells and prospects of its differentiation to oral epithelial equivalents.

OBJECTIVE: Although oral keratinocyte stem cells play a key role in tissue homeostasis, wound healing, and neoplasia, they remain difficult to identify and characterize. The specific aim of the present study is to characterize an oral keratinocyte stem-cell population separated using a magnetic technique. MATERIAL AND METHODS: Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using a proliferation-related marker, CD71 and α6ß4 integrin. The expression of different stem cell markers: CD44H, Nestin, Nanog, Oct 3÷4, CD117 was checked by immunofluorescence. The ability of α6ß4pos CD71neg fraction to form oral epithelial equivalents was also assayed. RESULTS: Three different oral keratinocyte subpopulations were obtained following magnetic separation: α6ß4pos CD71neg, α6ß4pos CD71pos and α6ß4neg. Our α6ß4pos CD71neg stem cell fraction was positive for Oct 3÷4, CD44H and cytokeratin 19 while Nanog, Nestin and CD117 expression was absent. At the same time, the other two cell fractions α6ß4pos CD71pos and α6ß4neg were negative for all stem cell markers. Also, α6ß4pos CD71neg fraction was able to regenerate a well stratified and organized oral epithelial equivalent. The distribution of cytokeratin 19 and involucrin in the oral epithelial equivalent reflected the in vivo situation in oral gingival epithelium. CONCLUSIONS: The human gingival α6ß4pos CD71neg fraction was strongly positive for a panel of stem cell markers and could form oral epithelial equivalent. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.

Magnetic separation and characterization of keratinocyte stem cells from human gingiva.

BACKGROUND AND OBJECTIVE: Although keratinocyte stem cells play a key role in tissue homeostasis, wound healing and neoplasia, they remain difficult to identify and characterize. The purpose of this study was to isolate and characterize an oral keratinocyte stem-cell population. MATERIAL AND METHODS: Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using α(6) ß(4) integrin and a proliferation-related marker, CD71. The isolated cell fractions were analyzed for cell size, cell cycle stage (using flow cytometry) and colony-forming ability. The expression of stem cell markers p63 and cytokeratin 19 and of differentiation markers cytokeratin 10 and involucrin was checked using immunocytochemical analysis. RESULTS: The stem cell CD71(neg) fraction had the smallest cell size compared with CD71(pos) and fractions [780.7 ± 141.5 (pixels), 1422.9 ± 264.6 (pixels) and 3844.4 ± 220.1 (pixels) respectively, p < 0.01; analysis of variance (ANOVA)]. Also, the CD71(neg) subpopulation consistently had the highest colony-forming ability among the three cell fractions (126.2 ± 21.7 vs. 32.8 ± 4.5 vs. 12.4 ± 2.1 compared with CD71(pos) and subpopulations, respectively, p < 0.01; ANOVA). Moreover, the CD71(neg) fraction contained more quiescent cells and fewer actively cycling cells than the CD71(pos) cell fraction. The candidate stem cells were positive for cytokeratin 19 and p63 keratinocyte stem cell markers, while differentiation markers such as cytokeratin 10 or involucrin were absent. CONCLUSION: The human gingival CD71(neg) cell fraction, separated by a magnetic system, demonstrated several characteristics of gingival keratinocyte stem cells. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.