Al-Bagdadi Fakhri A, Barona Humberto M, Martinez-Ceballos Eduardo, Yao Shaomian
Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.
Department of Mathematics, Southern University and A and M College, Baton Rouge, LA, USA.
J Microsc Ultrastruct. 2019 Apr-Jun;7(2):57-64. doi: 10.4103/JMAU.JMAU_44_18.
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.
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.
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.
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.
干细胞在组织更新和修复中发挥着重要作用。组织来源的干细胞已在组织工程和再生医学中得到应用。为了应用干细胞，需要通过培养将从组织中分离出的原代干细胞大量扩增。然而，已知组织干细胞在体外培养过程中通常会降低或丧失其干性特性。在本研究中，我们评估了大鼠牙囊干细胞（DFSCs）在体外培养过程中的超微结构变化。我们试图在超微结构水平上解释培养的组织干细胞干性特性的丧失。
从Sprague Dawley大鼠幼崽的第一磨牙中分离DFSCs，并在含有α-MEM加20%胎牛血清的培养基中培养。细胞在90%汇合时以1:3的比例传代，并在第3、6、7和9代收集，通过透射电子显微镜评估超微结构形态。
在所检查的四代（第3、6、7和9代）中，第3代粗面内质网（RER）扩张丰富，而第6、7和9代则较少。第3、7和9代扩张的RER中含有脂质。第3和6代的单核糖体和多核糖体位于线粒体和RER之间。第7代单核糖体和多核糖体丰富，尽管第9代主要是单核糖体。第3代膜结合的糖原颗粒存在于从细胞突出的液泡中。第9代一些糖原颗粒聚集在干细胞周边。第6、7和9代细胞核形状不规则且主要为常染色质。第9代线粒体暗且稀少；第7代不规则、小且暗；第6代小且呈圆形，它们在第3代中分散在远离细胞核的细胞质中。第6、7和9代可见细胞间接触。所检查的DFSCs的超微结构形态与未分化细胞的形态标准差异不大。第3代大液泡主要位于细胞周边，小液泡位于细胞中心。第6代小液泡散在细胞中心，较大的液泡在细胞周边观察到。
我们观察到以下超微结构变化：随着细胞传代次数增加，细胞精细的细胞质突起、扩张的细胞质液泡、细胞质吞饮小泡和核异染色质减少。相反，脂质球、核常染色质、不规则核形状以及细胞间接触的平均比例随传代次数增加。这些观察结果可能表明DFSCs长期培养后群体中定向分化细胞增加。
