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.

RARß2-dependent signaling represses neuronal differentiation in mouse ES cells.

Embryonic Stem (ES) cells are pluripotent cells that can be induced to differentiate into cells of all three lineages: mesoderm, endoderm, and ectoderm. In culture, ES cells can be differentiated into mature neurons by treatment with Retinoic Acid (RA) and this effect is mediated mainly through the activation of the RA nuclear receptors (RAR α, ß, and γ), and their isoforms. However, little is known about the role played by specific RAR types on ES cell differentiation. Here, we found that treatment of ES cells with AC55649, an RARß2 agonist, increased endodermal marker gene expression. On the other hand, we found that the inhibition of RARß with 5µM LE135, together with RA treatment, increased the efficiency of mouse ES cell differentiation into neurons by more than 4-fold as compared to cells treated with RA only. Finally, we performed proteomic analyses on ES cells treated with RA vs RA plus AC55649 in order to identify the signaling pathways activated by the RARß agonist. Our proteomic analyses using antibody microarrays indicated that proteins such as p38 and AKT were upregulated in cells treated with RA plus the agonist, as compared to cells treated with RA alone. Our results indicate that RARß may function as a repressor of neuronal differentiation through the activation of major cell signaling pathways, and that the pharmacological inhibition of this nuclear receptor may constitute a novel method to increase the efficiency of ES to neuronal differentiation in culture.

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.

Effect of the environmental pollutant hexachlorobenzene (HCB) on the neuronal differentiation of mouse embryonic stem cells.

Exposure to persistent environmental pollutants may constitute an important factor on the onset of a number of neurological disorders such as autism, Parkinson's disease, and Attention Deficit Disorder (ADD), which have also been linked to reduced GABAergic neuronal function. GABAergic neurons produce γ-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter in the brain. However, the lack of appropriate models has hindered the study of suspected environmental pollutants on GABAergic function. In this work, we have examined the effect of hexachlorobenzene (HCB), a persistent and bioaccumulative environmental pollutant, on the function and morphology of GABAergic neurons generated in vitro from mouse embryonic stem (ES) cells. We observed that: (1) treatment with 0.5 nM HCB did not affect cell viability, but affected the neuronal differentiation of ES cells; (2) HCB induced the production of reactive oxygen species (ROS); and (3) HCB repressed neurite outgrowth in GABAergic neurons, but this effect was reversed by the ROS scavenger N-acetylcysteine (NAC). Our study also revealed that HCB did not significantly interfere with the function of K+ ion channels in the neuronal soma, which indicates that this pollutant does not affect the maturation of the GABAergic neuronal soma. Our results suggest a mechanism by which environmental pollutants interfere with normal GABAergic neuronal function and may promote the onset of a number of neurological disorders such as autism and ADD.

All-trans-retinoid acid induces the differentiation of encapsulated mouse embryonic stem cells into GABAergic neurons.

Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that (1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and (2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (∼87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders.

Hoxa1 is required for the retinoic acid-induced differentiation of embryonic stem cells into neurons.

The ability of embryonic stem (ES) cells to differentiate into different cell fates has been extensively evaluated, and several protocols exist for the generation of various types of cells from mouse and human ES cells. We used a differentiation protocol that involves embryoid body formation and all-trans-retinoic acid (RA, 5 microM) treatment (EB/5 microM RA) to test the ability of Hoxa1 null ES cells to adopt a neuronal fate. Hoxa1(-/-) ES cells, when treated in this EB/5 microM RA protocol, failed to differentiate along a neural lineage; Hoxa1(-/-) ES cells express severalfold lower levels of many neuronal differentiation markers, including nestin, beta-tubulin III, and MAP2, and conversely, higher levels of endodermal differentiation markers (i.e., Sox17, Col4a1) than wild type (Wt) cells. Reintroduction of exogenous Hoxa1, under the control of the metallothionein I promoter, into Hoxa1(-/-) ES cells restored their capacity to generate neurons. Moreover, overexpression of Sox17, a gene that regulates endodermal differentiation, in Wt ES cells resulted in endodermal differentiation and in a complete abolition of beta-tubulin III expression. Thus, Hoxa1 activity is essential for the neuronal differentiation of ES cells in the presence of all-trans-RA, and Hoxa1 may promote neural differentiation by inhibiting Sox17 expression. Pharmacological manipulation of Hoxa1 levels may provide a method for promoting neuronal differentiation for therapeutic uses. Furthermore, because mutations in the Hoxa1 gene can cause autism spectrum disorder in humans, these data also provide important mechanistic insights into the early developmental processes that may result in this disorder.

Diepoxybutane activates the mitochondrial apoptotic pathway and mediates apoptosis in human lymphoblasts through oxidative stress.

Diepoxybutane (DEB) is the most potent metabolite of the environmental chemical 1,3-butadiene (BD), which is prevalent in petrochemical industrial areas. BD is a known mutagen and human carcinogen, and possesses multi-systems organ toxicity. We recently reported that DEB-induced cell death in TK6 lymphoblasts was due to the occurrence of apoptosis, and not necrosis. In this study, we investigated the molecular mechanisms responsible for DEB-induced apoptosis in these cells. Bax and Bak were found to be over-expressed and activated, and the mitochondrial trans-membrane potential was attenuated in cells undergoing DEB-induced apoptosis. Cytochrome c was depleted from the mitochondria of TK6 cells undergoing apoptosis, and was released into the cytosol in Jurkat T-lymphoblasts exposed to the same concentrations of DEB. Executioner caspase 3 was deduced to be activated by initiator caspase 9. DEB-induced reactive oxygen species (ROS) formation, and the ROS scavenger N-acetyl-L-cysteine effectively blocked DEB-induced apoptosis in TK6 cells. Collectively, these results demonstrate that the mitochondrial apoptotic pathway is activated to mediate DEB-induced apoptosis in human TK6 lymphoblasts. These results further demonstrate that DEB-induced apoptosis is also mediated by the DEB-induced generation of ROS. This is the first report to examine the mechanism of DEB-induced apoptosis in human lymphoblasts.

Differences in gene expression between wild type and Hoxa1 knockout embryonic stem cells after retinoic acid treatment or leukemia inhibitory factor (LIF) removal.

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of the Hoxa1 gene, the most 3' member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1(-/-) embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1(-/-) mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1(-/-) ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1, Postn/Osf2, and the bone sialoprotein gene or BSP), genes that are expressed in the developing brain (e.g. Nnat, Wnt3a, BDNF, RhoB, and Gbx2), and genes involved in various cellular processes (e.g. M-RAS, Sox17, Cdkn2b, LamA1, Col4a1, Foxa2, Foxq1, Klf5, and Igf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1, Oct3/4, Fgf4, and Bmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1(-/-) ES cells express high levels of various endodermal markers, including Gata4 and Dab2, and express much less Fgf5 after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.