Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Apr-Jun;7(2):57-64.
doi: 10.4103/JMAU.JMAU_44_18.

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

Affiliations

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

Fakhri A Al-Bagdadi et al. J Microsc Ultrastruct. 2019 Apr-Jun.

Abstract

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 in vitro 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.

Keywords: Dental follicle; mitochondria; nuclei; stem cells; transmission electron microscopy; ultrastructure.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts of interest.

Figures

Figure 1
Figure 1
Ultrastructure of Passage 3 dental follicle stem cells. It shows dilated rough endoplasmic reticulum, vacuoles (V), elongated mitochondria (white arrows), and abundant coarse microvilli (black arrows). N = Irregular nucleus with marginated heterochromatin. X 12,000. Scale bar: 2 μm
Figure 2
Figure 2
Ultrastructure of Passage 3 dental follicle stem cells. It shows dilated rough endoplasmic reticulum; some contain lipid (arrows). Scattered mitochondria of various shapes with distinct cristae (M) are also observed. X 24,900. Scale bar: 500 nm. 80 KV
Figure 3
Figure 3
Ultrastructure of Passage 3 dental follicle stem cells. It shows abundant rough endoplasmic reticulum, elongated mitochondria (arrow), cellular microvilli (arrows), and two irregular euchromatic nuclei (N) with narrow rims of heterochromatin. It is a cementoblast. X 1000
Figure 4
Figure 4
Ultrastructure of Passage 7 dental follicle stem cells. It shows dilated rough endoplasmic reticulum, mitochondria (M), irregular euchromatic nucleus (N), nucleoli (Ni), and dilated rough endoplasmic reticulum with lipid droplets (L). Cell junctions (arrows) are observed. X 19,200. Scale bar: 500 nm. 80 KV
Figure 5
Figure 5
Ultrastructure of Passage 9 dental follicle stem cells. It shows contact between cells (arrow) and nuclei (N) containing mainly euchromatin. Scattered vacuoles (V), several lipid globules (L), and mitochondria (M) are observed. X 10,000. Scale bar: 2 μm. 80 KV
Figure 6
Figure 6
Ultrastructure of Passage 6 dental follicle stem cells. It shows dilated rough endoplasmic reticulum (arrows), electron-dense mitochondria (M), irregular euchromatic nucleus (N) with two nucleoli (Ni), scattered vacuoles (V), and neighboring stem cell (black arrows). X 19,200. Scale bar: 2 μm. 80 KV
Figure 7
Figure 7
Ultrastructural cell characteristics whose mean ratios decreased with passage number. (a) Fine cell cytoplasmic processes. (b) Dilated cytoplasmic vacuoles. Decreased 16% for Passage 6, 45% for Passage 7, and 55% in Passage 9. (c) Cytoplasmic pinocytotic vesicles. (d) Nuclear heterochromatin
Figure 8
Figure 8
Ultrastructural cell characteristics whose mean ratios increased with passage number. (a) Lipid globules. Increases were observed in Passages 7 and 9. (b) Nuclear euchromatin. (c) Regular nuclear shape. (d) Cell contact between cells
Figure 9
Figure 9
Ultrastructure characteristics with irregular pattern. (a) Dilated rough endoplasmic reticulum profiles. (b) Irregular cell shape. (c) Mitochondrial density. (d) Golgi apparatus. Only observed in Passage 3 cells

Similar articles

Cited by

References

    1. Shanti RM, Li WJ, Nesti LJ, Wang X, Tuan RS. Adult mesenchymal stem cells: Biological properties, characteristics, and applications in maxillofacial surgery. J Oral Maxillofac Surg. 2007;65:1640–7. - PubMed
    1. Varga V, Hollý D, Vojtaššák J, Bohmer D, Polák S, Danišovič L. Morphological characterization of in vitro expanded human dental pulp-derived stem cells. Biol Sect Zool. 2011;66:706–11.
    1. d’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, et al. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: A pivotal synergy leading to adult bone tissue formation. Cell Death Differ. 2007;14:1162–71. - PubMed
    1. Kadar K, Kiraly M, Porcsalmy B, Molnar B, Racz GZ, Blazsek J, et al. Differentiation potential of stem cells from human dental origin – Promise for tissue engineering. J Physiol Pharmacol. 2009;60(Suppl 7):167–75. - PubMed
    1. Suchanek J, Soukup T, Visek B, Ivancakova R, Kucerova L, Mokry J, et al. Dental pulp stem cells and their characterization. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2009;153:31–5. - PubMed