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Differentiation of Stem Cells in the Dental Follicle

Department of Comparative Biomedical Sciences, Louisiana State University, School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70803, USA

View larger version (70K): [in this window] [in a new window]   Figure 1. DF cells grown in a stem cell medium form cell clusters after 2 wks of culture (A), and the clusters stained positive for alkaline phosphatase (B). In contrast, DF cells grown in the normal MEM medium did not form cell clusters, and no alkaline phosphatase staining was seen (C). (This Figure appears in color in the online version.)

View larger version (41K): [in this window] [in a new window]   Figure 2. Fluorescent staining of DF cells with Hoechst 33342. Note that the fluorescence staining of nuclei was weaker in some cells (arrows), putative stem cells, as compared with the majority with bright fluorescence (A). RT-PCR to detect gene expression of BCRP. Note that BCRP expression was detected in the DF of the rat first mandibular molar at post-natal days 5, 7, 9, and 11 in vivo, as well as in the cultured DF cells (B).

View larger version (101K): [in this window] [in a new window]   Figure 3. Differentiation of DF stem cells into various cell types. Alizarin red staining (A) and von Kossa staining (B) to determine osteogenic differentiation: The red staining in Alizarin red and black staining in von Kossa indicate the deposition of mineralization (A,B). No such staining was seen in the controls not subjected to osteogenic induction (C-a, control for Alizarin red staining; C-b, control for von Kossa staining). Oil Red O stain to detect adipogenesis (D). Note the stained adipocytes (arrow) in the induction treatment (D) vs. no adipocytes in the control without adipogenic induction (E). Multipolar neurons (arrow) were seen in the neuron induction treatment (F), while no neurons were seen in the control maintained in the stem cell medium (G). Immunostaining for neurofilament 200, a late neuron differentiation marker (H). Note that the heavy staining was seen in the induced neurons (H), while no staining appeared in the control cells maintained in the growth medium only (I). No immunostaining was seen when primary antibody was substituted with IgG in the immunostaining controls (J).

View larger version (123K): [in this window] [in a new window]   Figure 4. Treatment of the DF cells with doxorubicin (DOX). The number of surviving cells was greatly reduced as the duration of DOX (1 µM) incubation was increased from 2 hrs (B), to 4 hrs (C), and to 6 hrs (D), as compared with the control without DOX treatment (A). The placement of cells treated with DOX for 4 hrs in an adipogenesis induction medium resulted in the majority of the cells forming Oil Red O-positive adipocytes (arrow) (E), whereas in the controls not treated with DOX, but placed in adipogenesis induction medium, the majority of the cells remained undifferentiated, with only a few forming adipocytes (arrow) (F). The results suggest that DOX kills the non-stem cells, and that adipocytes are derived from the stem cells. (This Figure appears in color in the online version.)