Telomere Attrition Occurs during Ex Vivo Expansion of Human Dental Pulp Stem Cells

We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S) was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications

Endoglin co-expression with eNOS, SMAD2 and phosphorylated SMAD23 in normocholesterolemic and hypercholesterolemic mice: an immunohistochemical study

Endoglin, a homodimeric transmembrane glycoprotein, is a part of the transforming growth factorß (TGF-ß) receptor cascade. It has been demonstrated that endoglin can affect TGF-ß signaling and eNOS expression by affecting SMAD proteins in vitro. We planned to go one step forward and evaluate whether endoglin is co-expressed with SMAD2, phosphorylated SMAD2/3 protein and eNOS in endothelium of normocholesterolemic C57BL/6J mice, and in advanced atherosclerotic lesions in hypercholesterolemic apoE/LDLr-deficient mice by means of fluorescence immunohistochemistry. Female C57BL/6J mice were fed with a chow diet (standard laboratory diet) for 12 weeks after weaning (at the age of 4 weeks). Two-month-old female apoE/LDLrdeficient mice were fed the western type diet (atherogenic diet) containing 21% fat (11% saturated fat) and 0.15% cholesterol for 2 months. Immunohistochemical analysis of endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS expression was performed in mice aortic sinus. Immunohistochemical analysis showed the expression of endoglin in intact endothelium in both C57BL/6J and apoE/LDLr-deficient mice and in endothelium covering the atherosclerotic lesion in apoE/LDLr-deficient mice. Fluorescence immunohistochemistry revealed co-expression of endoglin with SMAD2, phosphorylated SMAD2/3 and eNOS in intact aortic endothelium in C57BL/6J mice. Moreover, strong co-localization of endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS was also detected in endothelium covering atherosclerotic lesions in apoE/LDLr-deficient mice. In conclusion, we suggest that endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS may be important in vessel endothelium homeostasis underlying their role in atherogenesis

Methotrexate released in vitro from bone cement inhibits human stem cell proliferation in S/G2 phase

Methotrexate (MTX) released from bone cement showed a useful local effect in animal models of bone tumours. However, local toxic reactions such as impaired wound healing were observed in areas surrounding the MTX-loaded implant. Therefore, we hypothesised that MTX released from bone cement would have harmful effects on human mesenchymal stem cells (MSC)—one of the basic components of bone marrow and tissue reparatory processes. Moreover, elution of MTX was calculated from implants prepared either with liquid or powdered MTX. During the 28-day incubation, the cement compounded with liquid MTX showed the highest elution rate of the drug. MTX released from pellets produced a significant decrease in proliferation of MSC as a consequence of a blockade of their cell cycle in the S/G2 phase. These findings indicate impairment of stem cell function in marginal areas surrounding the MTX-loaded cement and may help to explain problems with regeneration of tissues in these locations