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

Determining Direction of Axonal Flow in the Equine Ramus Communicans by Ultrastructural Examination of the Plantar Nerves 2 Months after Transecting the Ramus.

The ramus communicans, neural connection between medial and lateral plantar nerves of the horse, was transected to determine the degree to which medial and lateral plantar nerves contribute to the plantar ramus. After 2 months, sections of plantar nerves immediately proximal and distal to the communicating branch were collected and processed for electron microscopy. All examined nerves had undergone Wallerian degeneration and contained regenerating and mature fibers. Layers of the myelin sheath were separated by spaces and vacuoles, indicating demyelination of medial and lateral plantar nerves. Shrunken axons varied in diameter and were surrounded by an irregular axolemma. Shrunken axoplasm of both myelinated and non-myelinated fibers contained ruptured mitochondria and cristae, disintegrating cytoskeleton, and vacuoles of various sizes. The cytoplasm of neurolemmocytes contained various-sized vesicles, ruptured mitochondria within a fragile basal lamina and myelin whorls of multilayered structures indicative of Wallerian degeneration. These ultrastructural changes, found proximal and distal to the ramus in medial and lateral plantar nerves, suggest that axonal flow is bi-directional through the ramus communicans of the pelvic limbs of horses, a previously unreported finding. As well, maturity of nerves proximal and distal to the ramus indicates that all nerve fibers do not pass through the ramus.

Observation on the ultrastructure morphology of HeLa cells treated with ethanol: Statistical analysis.

It is estimated that 5.9% of all human deaths are attributable to alcohol consumption and that the harmful use of ethanol ranks among the top five risk factors for causing disease, disability, and death worldwide. Ethanol is known to disrupt phospholipid packing and promote membrane hemifusion at lipid bilayers. With the exception of mitochondria involved in hormone synthesis, the sterol content of mitochondrial membranes is low. As membranes that are low in cholesterol have increased membrane fluidity and are the most easily disordered by ethanol, we hypothesize that mitochondria are sensitive targets for ethanol damage. HeLa cells were exposed to 50 mM ethanol and the direct effects of ethanol on cellular ultrastructure were examined utilizing transmission electron microscopy. Our ultramicroscopic analysis revealed that cells exposed to ethanol harbor fewer incidence of apoptotic morphology; however, significant alterations to mitochondria and to nuclei occurred. We observed statistical increases in the amount of irregular cells and cells with multiple nuclei, nuclei harboring indentations, and nuclei with multiple nucleolus-like bodies. Indeed, our analysis revealed that mitochondrial damage is the most extensive type of cellular damage. Rupturing of cristae was the most prominent damage followed by mitochondrial swelling. Ethanol exposure also resulted in increased amounts of mitochondrial rupturing, organelles with linked membranes, and mitochondria localizing to indentations of nuclear membranes. We theorize that these alterations could contribute to cellular defects in oxidative phosphorylation and, by extension, the inability to generate regular levels of cellular adenosine triphosphate.

Morphometric and Ultrastructure studies of Primordial Follicles and Expression of Estrogen-dependent Genes in the Ovaries of Domestic Cats.

The aim of this study was to compare and estimate the population of the primordial follicle morphometrically and ultrastructurally in the left and right side ovaries of 10 ovariohysterectomied healthy domestic shorthair cats. The ovaries were processed for light microscopy, electron microscopy, and estrogen-dependent gene expression for assessments. A total of 15,092 primordial follicles with and without a nucleus were examined and counted. A total of 6842 primordial follicles with a nucleus were examined and counted. The light-microscopy numerical data were collected from two histological sections per ovary for a total of 20 sections from the left ovary and 20 sections from the right ovary. The average surface area of the histological sections was 645.99 mm2. The number of tertiary follicles was found to be higher in the left ovaries than in the right ovaries. The primordial follicles are under the tunica albuginea at various levels. Some are crowded or scattered in one or two rows, although at times, there were areas without any primordial follicles. The primordial follicles varied in size, and were surrounded by 4-10 squamous granulosa cells. Some primordial follicles shared their ooplasm with one or two neighboring primordial follicles, forming a giant primordial follicle with two or three nuclei. The ultrastructure of the primordial follicles showed rounded nuclei with distinct nucleoli, rounded and elongated mitochondria, and a considerably thick basement membrane under the granulosa cells. The squamous granulosa cells showed well-developed microvilli intermingled with the microvilli of the oocyte oolemma. Elongated mitochondria, coated pits, multicytoplasmic vesicles, ribosomes, and Golgi apparatuses were obvious in the oocyte ooplasm. Large vesicles contain small multivesicles and some scattered lipid globules in the ooplasm. There were estrogen-dependent gene-expression differences between the right and left ovaries. Further gene research is in the plan, using a larger pool of cats, with a focus on age differences.

Acute lymphoblast leukemia in sheep induced by inoculation of bovine leukemia virus: diagnostic electron microscopic morphological study.

Six control lambs were inoculated with Tris buffer, 7 lambs were inoculated with an early passage of bovine leukemia virus (B.L.V.) culture, and 7 lambs with a late passage B.L.V. All experimental lambs were positive with the agar gel immunodiffusion assay (AGID) within 3 months of inoculation and remained positive throughout the 8-year duration of the experiment. The earliest onset of leukemia was at 14 months and the latest was at 44 months after inoculation. Five lambs died with leukemia, two were inoculated with early passage, and three were inoculated with late passage of B.L.V. Eight years after the inoculation, the remaining nine inoculated lambs were clinically normal. The diagnostic ultrastructural morphology of the leukemic lymphoblasts in this study were characterized by hand-mirror cells, multiple nucleoli, irregular nuclear contour with deep indentions, electron-dense granules in the euchromatin, and nuclear cytoplasmic pockets, nuclear myelin figures, mitochondrial variation in size and density, disruption of rough endoplasmic reticulum, and increased ribosomal density. This study shows abundant cytoplasmic processes of hairy cell leukemia. The nuclei of the leukemia lymphoblasts showed electron-dense granules of varying sizes, which were not seen in any of the normal lymphoblasts.