Renza Spelat

Dental Pulp Stem Cells Differentiation Reveals New Insights in Oct4A Dynamics

Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.

Isolation and characterization of human dental pulp derived stem cells by using media containing low human serum percentage as clinical grade substitutes for bovine serum.

Adult stem cells have been proposed as an alternative to embryonic stem cells to study multilineage differentiation in vitro and to use in therapy. Current culture media for isolation and expansion of adult stem cells require the use of large amounts of animal sera, but animal-derived culture reagents give rise to some questions due to the real possibility of infections and severe immune reactions. For these reasons a clinical grade substitute to animal sera is needed. We tested the isolation, proliferation, morphology, stemness related marker expression, and osteoblastic differentiation potential of Dental Pulp Stem Cells (DPSC) in a chemically defined medium containing a low percentage of human serum, 1.25%, in comparison to a medium containing 10% Fetal Bovine Serum (FBS). DPSCs cultured in presence of our isolation/proliferation medium added with low HS percentage were obtained without immune-selection methods and showed high uniformity in the expression of stem cell markers, proliferated at higher rate, and demonstrated comparable osteoblastic potential with respect to DPSCs cultured in 10% FBS. In this study we demonstrated that a chemically defined medium added with low HS percentage, derived from autologous and heterologous sources, could be a valid substitute to FBS-containing media and should be helpful for adult stem cells clinical application.

Adipose tissue-derived stem cell in vitro differentiation in a three-dimensional dental bud structure.

Tooth morphogenesis requires sequential and reciprocal interactions between the cranial neural crest-derived mesenchymal cells and the stomadial epithelium, which regulate tooth morphogenesis and differentiation. We show how mesenchyme-derived single stem cell populations can be induced to transdifferentiate in vitro in a structure similar to a dental bud. The presence of stem cells in the adipose tissue has been previously reported. We incubated primary cultures of human adipose tissue-derived stem cells in a dental-inducing medium and cultured the aggregates in three-dimensional conditions. Four weeks later, cells formed a three-dimensional organized structure similar to a dental bud. Expression of dental tissue-related markers was tested assaying lineage-specific mRNA and proteins by RT-PCR, immunoblot, IHC, and physical-chemical analysis. In the induction medium, cells were positive for ameloblastic and odontoblastic markers as both mRNAs and proteins. Also, cells expressed epithelial, mesenchymal, and basement membrane markers with a positional relationship similar to the physiologic dental morphogenesis. Physical-chemical analysis revealed 200-nm and 50-nm oriented hydroxyapatite crystals as displayed in vivo by enamel and dentin, respectively. In conclusion, we show that adipose tissue-derived stem cells in vitro can transdifferentiate to produce a specific three-dimensional organization and phenotype resembling a dental bud even in the absence of structural matrix or scaffold to guide the developmental process.

Serine 111 Phosphorylation Regulates OCT4A Protein Subcellular Distribution and Degradation

Background: Self-renewal properties are attributed to critical amounts of the OCT4A transcription factor, and little is known about its post-translational regulation. Results: OCT4A interacts with ERK1/2 and is phosphorylated at Ser-111, increasing its ubiquitination and degradation. Discussion: These results suggest an increase in OCT4A degradation downstream of MEK1 activation and FGF2 treatment. Significance: Controlling the mechanism by which cells balance self-renewal would advance our knowledge of stem cells. Embryonic stem cell self-renewal properties are attributed to critical amounts of OCT4A, but little is known about its post-translational regulation. Sequence analysis revealed that OCT4A contains five putative ERK1/2 phosphorylation sites. Consistent with the hypothesis that OCT4A is a putative ERK1/2 substrate, we demonstrate that OCT4A interacts with ERK1/2 by using both in vitro GST pulldown and in vivo co-immunoprecipitation assays. MS analysis identified phosphorylation of OCT4A at Ser-111. To investigate the possibility that ERK1/2 activation can enhance OCT4A degradation, we analyzed endogenous ubiquitination in cells transfected with FLAG-OCT4A alone or with constitutively active MEK1 (MEK1CA), and we observed that the extent of OCT4 ubiquitination was clearly increased when MEK1CA was coexpressed and that this increase was more evident after MG132 treatment. These results suggest an increase in OCT4A ubiquitination downstream of MEK1 activation, and this could account for the protein loss observed after FGF2 treatment and MEK1CA transfection. Understanding and controlling the mechanism by which stem cells balance self-renewal would substantially advance our knowledge of stem cells.

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis. In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10−7M and 10−8M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains. In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. −90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules. Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.

Loss of Parafollicular Cells during Gravitational Changes (Microgravity, Hypergravity) and the Secret Effect of Pleiotrophin

It is generally known that bone loss is one of the most important complications for astronauts who are exposed to long-term microgravity in space. Changes in blood flow, systemic hormones, and locally produced factors were indicated as important elements contributing to the response of osteoblastic cells to loading, but research in this field still has many questions. Here, the possible biological involvement of thyroid C cells is being investigated. The paper is a comparison between a case of a wild type single mouse and a over-expressing pleiotrophin single mouse exposed to hypogravity conditions during the first animal experiment of long stay in International Space Station (91 days) and three similar mice exposed to hypergravity (2Gs) conditions. We provide evidence that both microgravity and hypergravity induce similar loss of C cells with reduction of calcitonin production. Pleiotrophin over-expression result in some protection against negative effects of gravity change. Potential implication of the gravity mechanic forces in the regulation of bone homeostasis via thyroid equilibrium is discussed.

Thyroid Cell Growth: Sphingomyelin Metabolism as Non-Invasive Marker for Cell Damage Acquired during Spaceflight

Prolonged spaceflights are known to elicit changes in human cardiovascular, musculoskeletal, and nervous systems, whose functions are regulated by the thyroid gland. It is known that sphingomyelin metabolism is involved in apoptosis (programmed cell death) of thyroid cells induced by UVC radiation, but at present no data exists with regard to this phenomenon, which occurs during space missions. The aim of this study was to analyze, for the first time, the effect of spaceflight on the enzymes of sphingomyelin metabolism, sphingomyelinase, and sphingomyelin synthase, and to determine whether the ratio between the two enzymes might be used as a possible marker for thyroid activity during space missions. Both quiescent thyroid cells and thyroid cells stimulated to proliferate with thyrotropin (TSH) were cultured during the Eneide and Esperia missions on the International Space Station. The results show that during space missions the cells treated with TSH grew only 1.5 ± 0.65-fold and, thus, behave similarly to quiescent cells, while on the ground the same cells, maintained in experimental conditions that reproduced those of the flight, grew 7.71 ± 0.67-fold. Comparison of the sphingomyelinase/sphingomyelin-synthase ratio and the levels of Bax, STAT3, and RNA polymerase II in proliferating, quiescent, pro-apoptotic, or apoptotic cells demonstrated that thyroid cells during space missions were induced into a pro-apoptotic state. Given its specificity and the small amount of cells needed for analysis, we propose the use of the sphingomyelinase/sphingomyelin-synthase ratio as a marker of functional status of thyroid cells during space missions. Further studies could lead to its use in real time during prolonged spaceflights.

Observing the Mouse Thyroid Sphingomyelin Under Space Conditions: A Case Study from the MDS Mission in Comparison with Hypergravity Conditions

This is a case report of apparent thyroid structural and functional alteration in a single mouse subjected to low Earth orbit spaceflight for 91 days. Histological examination of the thyroid gland revealed an increase in the average follicle size compared to that of three control animals and three animals exposed to hypergravity (2g) conditions. Immunoblotting analysis detected an increase in two thyroid gland enzymes, sphingomyelinase and sphingomyelin-synthase1. In addition, sphingomyelinase, an enzyme confined to the cell nucleus in the control animals, was found in the mouse exposed to hypogravity to be homogeneously distributed throughout the cell bodies. It represents the first animal observation of the influence of weightlessness on sphingomyelin metabolism.

The thyroid lobes: The different twins

Although differences in size of the right and left thyroid lobes are well defined, differences in morphology, follicles structure, cAMP production, thyrotropin receptor, and protein involved in cell signalling have not previously been reported. This study provides morpho-functional data of right and left thyroid lobes by biochemical, immunohistochemistry, immunoblotting and immunofluorescence analysis. We demonstrate that, in comparison with the left lobe, the right lobe has a higher activation index, is more sensitive to thyrotropin treatment, is rich in thyrotropin receptor and caveolin 1 involved in thyroid hormone synthesis as well as in epithelial thyroid cell homeostasis, is characterised by a high content of molecules involved in cell signalling such as stat3, raf1, sphingomyelinase and sphingomyelin-synthase whose activity ratio is necessary for epithelial cell activity and finally has more areas calcitonin-dependent. The relation between structure/function of right lobe and its susceptibility to the higher risk of pathological modifications with respect the left lobe is discussed.

Cultured Human Keratocytes from the Limbus and Cornea Both Express Epithelial Cytokeratin 3: Possible Mesenchymal-Epithelial Transition

The corneal limbus is the repository of epithelial stem cells (SC) that sustain the turnover of corneal epithelial cells. The limbus stroma contains mesenchymal SC that generates stromal keratocytes. Mesenchymal-epithelial transition is a phenomenon wherein cells of mesenchymal phenotype can transdifferentiate to epithelial phenotype. Our aim was to study whether limbal keratocytes, cytokeratin 3 (CK3) negative, could be induced to transdifferentiate into CK3 positive cells. Human keratocytes were isolated from the limbus and cornea of cadaver donors, cultured and evaluated for CD34, CK3 and vimentin expression by immunofluorescence and RTPCR and for keratocan by RT-PCR. All cells regardless of site expressed vimentin and some also expressed CD34 and CK3. Double immunofluorescence revealed three subpopulations: CK3−/CD34+, CK3+/CD34+ and CK3+/CD34−. Total CD34 cell yield was higher in the limbus with a peak time to confluence (TTC) of more than 30 days. Total CK3 cell yield was greater in the cornea with a peak TTC of less than 30 days. Increasing donor age corresponded to a decreased CD34 yield and an increased CK3 yield. CK3−/CD34+ and CK3+/CD34− cells behaved similarly to total CD34 and CK3 cells in relation to age, site and TTC while CK3+/CD34+ cells showed intermediate features. Keratocan was present in corneal samples.