Ultrastructure Morphological Characterization of Different Passages of Rat Dental Follicle Stem Cells at Culture.

Introduction: Stem cells play important roles in tissue renewal and repair. Tissue-derived stem cells have been demonstrated for their applications in tissue engineering and regenerative medicine. Expansion of primary stem cells isolated from tissues to a large quantity through culture is needed for application of the stem cells. However, it is known that tissue stem cells commonly reduce or lose their stemness properties during in vitro culture. In this study, we assessed ultrastructural changes of rat dental follicle stem cells (DFSCs) during in vitro culture. It is our attempt to explain the loss of stemness properties in cultured tissue-stem cells at the ultrastructural level.

Method: DFSCs was isolated from first molars of Sprague Dawley rat pups and cultured in medium consisting of alpha-MEM plus 20% FBS. Cells were passaged at 1 to 3 ratio at 90% confluence, and collected at passages 3, 6, 7 and 9 for assessment of ultrastructure morphology by transmission electron microscopy.

Results: Of the four passages (3, 6, 7, and 9) examined, dilated rough endoplasmic reticulum (RER) was abundant in Passage 3 but less so in Passages 6, 7, and 9. The dilated RER contained lipid in Passages 3, 7, and 9. The mono- and polyribosomes in Passages 3 and 6 were located between the mitochondria and the RER. Mono- and polyribosomes were abundant in Passage 7, although mainly monoribosomes were present in Passage 9. Membrane-bound glycogen granules were in vacuoles bulging off the cells in Passage 3. Some glycogen granules were grouped in the periphery of a stem cell in Passage 9. Nuclei shapes were irregular and mainly euchromatic in Passages 6, 7, and 9. The mitochondria were dark and scarce in Passage 9; irregular, small, and dark in Passage 7; and small and rounded in Passage 6, and they were spread in the cytoplasm away from the nucleus in Passage 3. Cell contacts were seen in Passages 6, 7, and 9. The ultrastructure morphology of the examined DFScs was not very different from the morphology criteria of the undifferentiated cells. Large vacuoles in Passage 3 were mainly at the periphery of the cell, with the small vacuoles in the cell center. Small vacuoles were scattered in the cell center of Passage 6 and the larger ones were observed at the cell's periphery.

Conclusions: We observed the following ultrastructural changes: decreases of fine cell cytoplasmic processes, dilated cytoplasmic vacuoles, cytoplasmic pinocytotic vesicles, and nuclear heterochromatin with increasing cell passage number. Conversely, mean ratios of lipid globules, nuclear euchromatin, irregular nuclear shape, and cell contact between cells were increased with passage number. The observations may suggest an increase in committed cells among the population after long-term culture of DFSCs.