Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831)

Abstract: The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin, and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

The BTB/POZ Transcription Factor Miz-1 Is Required To Regulate The Commitment, Survival And Differentiation Of Early B And T Cell Lineages

Les lymphocytes B et T sont issus de cellules progénitrices lymphoïdes de la moelle osseuse qui se différencient grâce à l’action de facteurs de transcription, cytokines et voies de signalisation, dont l’interleukine-7 (IL-7)/IL-7 récepteur (IL-7R). Le facteur de transcription c-Myc est exprimé par les cellules lymphoïdes et contrôle leur croissance et leur différenciation. Cette régulation transcriptionnelle peut être coordonnée par le complexe c-Myc/Myc-Interacting Zinc finger protein-1 (Miz-1). Le but de ce projet était de comprendre les mécanismes qui impliquent Miz-1 et le complexe c-Myc/Miz-1 dans le développement des lymphocytes B et T. Pour réaliser ce projet, des souris déficientes pour le domaine de transactivation de Miz-1 (Miz-1POZ) et des souris à allèles mutantes pour c-MycV394D, mutation qui empêche l’interaction avec Miz-1, ont été générées. La caractérisation des souris Miz 1POZ a démontré que l’inactivation de Miz-1 perturbe le développement des lymphocytes B et T aux stades précoces de leur différenciation qui dépend de l’IL-7. L’analyse de la cascade de signalisation IL-7/IL-7R a montré que ces cellules surexpriment la protéine inhibitrice SOCS1 qui empêche la phosphorylation de STAT5 et perturbe la régulation à la hausse de la protéine de survie Bcl-2. De plus, Miz-1 se lie directement au promoteur de SOCS1 et contrôle son activité. En plus de contrôler l’axe IL-7/IL-7R/STAT5/Bcl-2 spécifiquement aux stades précoces du développement afin d’assurer la survie des progéniteurs B et T, Miz-1 régule l’axe EBF/Pax-5/Rag-1/2 dans les cellules B afin de coordonner les signaux nécessaires pour la différenciation des cellules immatures. La caractérisation des souris c-MycV394D a montré, quant à elle, que les fonctions de Miz-1 dans les cellules B et T semblent indépendantes de c-Myc. Les cellules T des souris Miz-1POZ ont un défaut de différenciation additionnel au niveau de la -sélection, étape où les signaux initiés par le TCR remplacent ceux induits par IL-7 pour assurer la prolifération et la différenciation des thymocytes en stades plus matures. À cette étape du développement, une forme fonctionnelle de Miz-1 semble être requise pour contrôler le niveau d’activation de la voie p53, induite lors du processus de réarrangement V(D)J du TCR. L’expression de gènes pro-apoptotiques PUMA, NOXA, Bax et du régulateur de cycle cellulaire p21CIP1 est régulée à la hausse dans les cellules des souris Miz-1POZ. Ceci provoque un débalancement pro-apoptotique qui empêche la progression du cycle cellulaire des cellules TCR-positives. La survie des cellules peut être rétablie à ce stade de différenciation en assurant une coordination adéquate entre les signaux initiés par l’introduction d’un TCR transgénique et d’un transgène codant pour la protéine Bcl-2. En conclusion, ces études ont montré que Miz-1 intervient à deux niveaux du développement lymphoïde: l’un précoce en contrôlant la signalisation induite par l’IL-7 dans les cellules B et T, en plus de l’axe EBF/Pax-5/Rag-1/2 dans les cellules B; et l’autre tardif, en coordonnant les signaux de survie issus par le TCR et p53 dans les cellules T. Étant donné que les thymocytes et lymphocytes B immatures sont sujets à plusieurs rondes de prolifération, ces études serviront à mieux comprendre l’implication des régulateurs du cycle cellulaire comme c-Myc et Miz-1 dans la génération des signaux nécessaires à la différenciation non aberrante et à la survie des ces cellules. Enfin, les modèles expérimentaux, souris déficientes ou à allèles mutantes, utilisés pour ce travail permettront de mieux définir les bases moléculaires de la transformation maligne des lymphocytes B et T et de révéler les mécanismes conduisant au lymphome.

Bone marrow cells differentiation to neurons in the rat brain using Serotonin and GABA:Their role on glutamate receptors, IP3, cAMP and cGMP functional regulation in unilateral Parkinsonism induced by 6-hydroxydopamine

Parkinson's disease is a chronic progressive neurodegenerative movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Our findings demonstrated that glutamatergic system is impaired during PD. The evaluations of these damages have important implications in understanding the molecular mechanism underlying motor, cognitive and memory deficits in PD. Our results showed a significant increase of glutamate content in the brain regions of 6- OHDA infused rat compared to control. This increased glutamate content caused an increase in glutamatergic and NMDA receptors function. Glutamate receptor subtypes- NMDAR1, NMDA2B and mGluR5 have differential regulatory role in different brain regions during PD. The second messenger studies confirmed that the changes in the receptor levels alter the IP3, cAMP and cGMP content. The alteration in the second messengers level increased the expression of pro-apoptotic factors - Bax and TNF-α, intercellular protein - α-synuclein and reduced the expression of transcription factor - CREB. These neurofunctional variations are the key contributors to motor and cognitive abnormalities associated with PD. Nestin and GFAP expression study confirmed that 5-HT and GABA induced the differentiation and proliferation of the BMC to neurons and glial cells in the SNpc of rats. We also observed that activated astrocytes are playing a crucial role in the proliferation of transplanted BMC which makes them significant for stem cell-based therapy. Our molecular and behavioural results showed that 5-HT and GABA along with BMC potentiates a restorative effect by reversing the alterations in glutamate receptor binding, gene expression and behaviour abnormality that occur during PD. The therapeutic significance in Parkinson’s disease is of prominence.

Calcium Hydroxide Promotes Cementogenesis and Induces Cementoblastic Differentiation of Mesenchymal Periodontal Ligament Cells in a CEMP1-and ERK-Dependent Manner

Periodontal tissue engineering is a complex process requiring the regeneration of bone, cementum, and periodontal ligament (PDL). Since cementum regeneration is poorly understood, we used a dog model of dental pulpal necrosis and in vitro cellular wounding and mineralization assays to determine the mechanism of action of calcium hydroxide, Ca(OH)(2), in cementogenesis. Laser capture microdissection (LCM) followed by qRT-PCR were used to assay responses of periapical tissues to Ca(OH)(2) treatment. Additionally, viability, proliferation, migration, and mineralization responses of human mesenchymal PDL cells to Ca(OH)(2) were assayed. Finally, biochemical inhibitors and siRNA were used to investigate Ca(OH)(2)-mediated signaling in PDL cell differentiation. In vivo, Ca(OH)(2)-treated teeth formed a neocementum in a STRO-1- and cementum protein-1 (CEMP1)-positive cellular environment. LCM-harvested tissues adjacent to the neocementum exhibited higher mRNA levels for CEMP1, integrin-binding sialoprotein, and Runx2 than central PDL cells. In vitro, Ca(OH)(2) and CEMP1 promoted STRO-1-positive cell proliferation, migration, and wound closure. Ca(OH)(2) stimulated expression of the cementum-specific proteins CEMP1 and PTPLA/CAP in an ERK-dependent manner. Lastly, Ca(OH)(2) stimulated mineralization by CEMP1-positive cells. Blocking CEMP1 and ERK function abolished Ca(OH)(2)-induced mineralization, confirming a role for CEMP1 and ERK in the process. Ca(OH)(2) promotes cementogenesis and recruits STRO-1-positive mesenchymal PDL cells to undergo cementoblastic differentiation and mineralization via a CEMP1- and ERK-dependent pathway.

Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor, Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as rvell as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin, In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody, To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed, Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1, They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen, Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Revisiting AFLP fingerprinting for an unbiased assessment of genetic structure and differentiation of taurine and zebu cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Epigallocatechin-3-gallate induces cell death in acute myeloid leukaemia cells and supports all-trans retinoic acid-induced neutrophil differentiation via death-associated protein kinase 2

Acute promyelocytic leukaemia (APL) patients are successfully treated with all-trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin-3-gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG-induced cell death in acute myeloid leukaemia (AML). A significant increase of death-associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG-mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG-induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.

Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

Neuronal differentiation by indomethacin and IBMX inhibits proliferation of small cell lung cancer cells in vitro

Small cell lung cancer (SCLC) is one of the most aggressive malignancies implying a very poor prognosis for patients even under therapy. Since it is known that SCLC cells exhibit neurone-like characteristics, we investigated whether a neuronal induction medium (NID) consisting of indomethacin (200 ?M), 3-isobutyl-1-methylxanthine (IBMX, 500 ?M) and insulin (5 ?g/ml) induces neuronal differentiation and by this reduces malignancy of SCLC in vitro.

Deregulated ephrin-B2 signaling in mammary epithelial cells alters the stem cell compartment and interferes with the epithelial differentiation pathway

Cancer most probably originates from stem/progenitor cells and exhibits a similar cell hierarchy as normal tissues. Moreover, there is growing evidence that only the stem cells are capable of metastasis formation. We have previously shown that overexpression of a dominant negative ephrin-B2 mutant interferes with mammary gland differentiation and confers a metastatic phenotype to NeuT-induced mammary tumors with an increase in cells with stem/progenitor characteristics. To investigate the role of ephrin-B2 in the control of the mammary stem cell niche, we analyzed the mammary stem and progenitor cell populations in transgenic mice overexpressing the mutant ephrin-B2. Quantification by FACS analysis revealed a significant increase of cells in the basal/alveolar cell-, the bi-potent progenitor- and the stem cell-enriched fractions. Moreover, the supposed precursors of estrogen receptor-positive cells were elevated in the stem cell-enriched fraction. In contrast, the epithelium from transgenic mice overexpressing the native ephrin-B2 gene showed an augmentation of the luminal cell- and the bi-potent progenitor-enriched fractions. Repopulation assays revealed that the epithelial cells of truncated ephrin-B2 transgenic epithelial cells have a higher regeneration capacity than those of controls and of native ephrin-B2 transgenic mice, confirming the augmentation of stem cells. Morphologically, these outgrowths exhibited impaired basal/luminal compartmentalization and epithelial polarization. These results demonstrate that deregulated ephrin-B2 expression interferes with the regulation of the stem cell niche and leads to a shift of the differentiation pathway and may thereby contribute to the acquisition of the metastatic phenotype long before carcinogenic growth becomes apparent.

Acid and alkali etching of grit blasted zirconia: Impact on adhesion and osteogenic differentiation of MG63 cells in vitro

There is a need for evaluating zirconia surface modifications and their potential impact on the biological response of osteogenic cells. Grit blasted zirconia discs were either left untreated or underwent acid or alkaline etching. Adhesion and osteogenic differentiation of MG63 cells was determined after one week of culture. The macro-scaled roughness of the grit blasted zirconia discs, independent of the surface treatment, was within a narrow range and only slightly smoother than titanium discs. However, the alkaline- and acid-etching led to an increase of the micro-roughness of the surface. The surface modifications had no effect on cell spreading and did not cause significant change in the expression of differentiation markers. Thus, in this respective setting, morphologic changes observed upon treatment of grit blasted zirconia discs with acid or alkaline do not translate into changes in MG63 cell adhesion or differentiation and are comparable to findings with anodized titanium discs.