Differentiation capacity of mouse dental pulp stem cells into osteoblasts and osteoclasts.

OBJECTIVE: Our research attempted to show that mouse dental pulp stem cells (DPSCs) with characters such as accessibility, propagation and higher proliferation rate can provide an improved approach for generate bone tissues. With the aim of finding and comparing the differentiation ability of mesenchymal stem cells derived from DPSCs into osteoblast and osteoclast cells; morphological, molecular and biochemical analyses were conducted. MATERIALS AND METHODS: In this experimental study, osteoblast and osteoclast differentiation was induced by specific differentiation medium. In order to induce osteoblast differentiation, 50 µg mL(-1) ascorbic acid and 10 mM ß-glycerophosphate as growth factors were added to the complete medium consisting alpha-modified Eagle's medium (α-MEM), 15% fetal bovine serum (FBS) and penicillin/streptomycin, while in order to induce the osteoclast differentiation, 10 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL) and 5 ng/mL macrophage-colony stimulating factor (M-CSF) were added to complete medium. Statistical comparison between the osteoblast and osteoclast differentiated groups and control were carried out using t test. RESULTS: Proliferation activity of cells was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Statistical results demonstrated significant difference (p<0.05) between the control and osteoblastic induction group, whereas osteoclast cells maintained its proliferation rate (p>0.05). Morphological characterization of osteoblast and osteoclast was evaluated using von Kossa staining and May-Grunwald-Giemsa technique, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) molecular analysis demonstrated that mouse DPSCs expressed Cd146 and Cd166 markers, but did not express Cd31, indicating that these cells belong to mesenchymal stem cells. Osteoblast cells with positive osteopontin (Opn) marker were found after 21 days, whereas this marker was negative for DPSCs. CatK, as an osteoclast marker, was negative in both osteoclast differentiation medium and control group. Biochemical analyses in osteoblast differentiated groups showed alkaline phosphatase (ALP) activity significantly increased on day 21 as compared to control (p<0.05). In osteoclast differentiated groups, tartrate-resistant acid phosphatase (TRAP) activity representing osteoclast biomarker didn't show statistically significant as compared to control (p>0.05). CONCLUSION: DPSCs have the ability to differentiate into osteoblast, but not into osteoclast.

Differentiation of dental pulp stem cells into neuron-like cells in serum-free medium.

Dental pulp tissue contains dental pulp stem cells (DPSCs). Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells) are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146(+) , Cd166(+) , and Cd31(-) in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

In vitro chondrogenesis transformation study of mouse dental pulp stem cells.

A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC) and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.). Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P < 0.05). Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P < 0.05) in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.

Characterization of mononucleated human peripheral blood cells.

Unspecialized cells that can renew themselves and give rise to multiple differentiated cell types are termed stem cells. The objective of this study was to characterize and investigate, through molecular and biochemical analyses, the stemness of cells derived from isolated mononucleated cells that originated from peripheral blood. The isolated mononucleated cells were separated according to their physical characteristics (adherent and suspension), after 4 to 7 days into a 14-day culturing period in complete medium. Our results revealed that adherent and suspension cells were positive for mesenchymal stem cell (MSC) and hematopoietic stem cell (HSC) markers, respectively. Differentiation of adherent cells into osteoblasts was associated with expression of the OPN gene and increasing ALP enzyme activity, while differentiation of suspension cells into osteoclasts was associated with expression of the TRAP gene and increasing TRAP enzyme activity. In conclusion, molecular and biochemical analyses showed that mononucleated cells consist of MSC (adherent) and HSC (suspension), and both cell types are able to differentiate into specialized cells from their respective lineage: osteoblast (MSC) and osteoclast (HSC).

Effect of lead on proliferation and neural differentiation of mouse bone marrow-mesenchymal stem cells.

Bone marrow-mesenchymal stem cells (MSCs) are considered to be an ideal source of stem cells for assessing the effects of environmental toxins on the proliferation, multipotency and differentiation of adult stem cells. The aim of this study was to investigate the effect of lead on the proliferation and neuronal differentiation of murine MSCs. MTT assay used in this study revealed that while the proliferation of MSCs is sensitive to higher than 10 microM lead, a 50% reduction in the rate of their proliferation can be achieved in the presence of 60 microM lead. The results of immunocytochemistry and RT-PCR showed that beta-mercaptoethanol induced-neuronal differentiation is also reduced after the treatment of MSCs by 60 microM lead. Furthermore, the comet assay analysis of MSCs showed a substantial increase in DNA damage in the lead treated cells compared to the control. In conclusion our results revealed for the first time that lead is not only cytotoxic to the survival and proliferation of MSCs but also inhibits their differentiation to neurons in a dose-dependant manner. Therefore, MSCs appear to be an alternative method for assessing the cytotoxic effects of such environmental hazards.