Drifter technique: a new method to obtain metaphases in Hep-2 cell line cultures

The Hep-2 cell line is derived from laryngeal carcinoma cells and is often utilized as a model in carcinogenesis and mutagenesis tests. To evaluate the proliferative potential of this line, we developed a cytogenetic methodology (drifter technique) to obtain metaphases from cells that loose cellular adhesion when they underwent mitosis in culture. By this procedure, 2000 cells were counted, resulting in a mitotic index (MI) of 22.2%. Although this MI was not statistically different from the one obtained using either a classical cytogenetic method or a cell synchronization technique, the drifter technique has the advantage of not requiring the use of some reagents for the obtention of metaphases and also of diminishing the consumption of maintenance reagents for this cell line.A linhagem celular Hep-2 é formada por células de carcinoma da laringe e é muito utilizada em modelos de carcinogênese e mutagenêse. Para avaliar o potencial proliferativo desta linhagem, desenvolvemos uma metodologia citogenética (técnica do sobrenadante) para obtenção de metáfases a partir de células que, ao entrarem em mitose, perdem adesão celular, ficando em suspensão no meio de cultura. Através deste procedimento, foram contadas 2000 células, correspondendo a um índice mitótico (IM) de 22.2% . Apesar de o IM obtido por esta técnica não ter sido estatisticamente diferente do IM obtido por outras metodologias citogenéticas clássicas, a técnica do sobrenadante é vantajosa porque elimina o uso de alguns reagentes utilizados na obtenção de metáfases e também diminui o consumo de reagentes de manutenção desta linhagem.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal do Pará Centro de Ciências Biológicas Departamento de BiologiaUniversidade de São Paulo Faculdade de Medicina de Ribeirão Preto Departamento de GenéticaUniversidade Federal do Pará Centro de Ciências Biológicas Departamento de GenéticaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MorfologiaUNIFESP, EPM, Depto. de MorfologiaSciEL

Análise de células-tronco adultas (CTA) em cultura de células de tecido epitelial de pequenos roedores (rodentia-stricognathi- sciurognathi)

The Adult Stem Cells (ASC) are non-specialized multipotent cells found in the bone marrow, peripheral blood, cornea, retina, brain, muscles, dental pulp, liver, pancreas, skin epithelium, digestive system, umbilical cord and placenta. These cells can indefinably reproduce and renew themselves and, under some stimulation, to change into specialized cells of different tissues or organs. The present work had the aim of obtaining ASC from epithelial tissues from wild rodents of different species (Oecomys concolor – one female, Proechimys roberti – two males, Hylaeamys megacephalus – two males). The methodology for isolation and in vitro culture of epithelial tissue following the previously described protocols, as well as the analysis after cryopreservation of morphology, genome stability, counting and cells viability, clonogenic potential and differentiation on osteocytes, chondrocytes and adipocytes. The ADC were characterized as a homogeneous population of in vitro growing cells adherent to plastic surfaces, which has a morphology similar to fibroblasts and with fusiform shape, with high growing rate and cell proliferation form many successive passages, where the clonogenic assays evaluated the cell renewing. On checking the genome stability on P3, the entire sample had stable karyotypes with the correct diploid number. The methodology for ASC differentiation into osteocytes, chondrocytes and adipocytes cell lines was satisfactory and the cells demonstrated the staining with Alizarin Red S, Alcian Blue and Oil Red O, respectively. The entire sample had capacity of proliferation and differentiation, being a potential source of skin ASC. These species can be used as models for ASC studies.FAPESPA - Fundação Amazônia de Amparo a Estudos e PesquisasCNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoAs células-tronco adultas (CTA) são células multipotentes e não especializadas encontradas na medula óssea, no sangue periférico, na córnea, na retina, no cérebro, no músculo esquelético, na polpa dental, no fígado, no pâncreas, no epitélio da pele, no sistema digestivo, no cordão umbilical e na placenta. Estas células podem se renovar e reproduzir indefinidamente e, sob certos estímulos, se transformar em células especializadas de diferentes tecidos ou órgãos. O presente trabalho tem como objetivo a obtenção de CTA a partir de tecido epitelial de roedores silvestres de espécies diferentes (Oecomys concolor - um exemplar fêmea, Proechimys roberti - dois exemplares machos, Hylaeamys megacephalus - dois exemplares machos). A metodologia para isolamento e cultivo in vitro de amostras do tecido epitelial foi estabelecida, a partir de protocolos já descritos, avaliando aspectos morfológicos, estabilidade genômica, contagem e análise da viabilidade celular, potencial clonogênico e indução de diferenciação em osteócitos, condrócitos e adipócitos. Todas essas análises foram feitas pós-criopreservação das culturas. As CTA foram caracterizadas como população homogênea de células que proliferam in vitro, como células aderentes à superfície do plástico, tendo morfologia semelhante a fibroblastos e formato fusiforme, com alta taxa de crescimento e proliferação celular por várias passagens sucessivas, onde a autorrenovação celular foi avaliada por ensaios clonogênicos. Na análise para examinar a estabilidade genômica na P3, todas as amostras apresentaram cariótipo com número diplóide normal e estável. A metodologia empregada nos ensaios para diferenciação das CTA em linhagens osteogênica, condrogênica e adipogênica, apresentou resultados satisfatórios, onde as células mostraram a marcação desejada através das colorações Alizarin Red S, Alcian Blue e Oil Red O, respectivamente. Todas as amostras testadas apresentam capacidade de proliferação e diversidade de diferenciação, sendo potencialmente fornecedores de CTA provenientes da pele e podendo ser utilizados como organismos modelos de estudos em CT

Chromosomal studies in Callicebus donacophilus pallescens, with classic and molecular cytogenetic approaches: multicolour FISH using human and Saguinus oedipus painting probes

Universidade Federal do Pará. Centro de Ciências Biológicas. Departamento de Genética. Belém, PA, Brasil.Universidade Federal do Pará. Centro de Ciências Biológicas. Departamento de Genética. Belém, PA, Brasil.Universidade Federal do Pará. Centro de Ciências Biológicas. Departamento de Genética. Belém, PA, Brasil.Universidade Federal do Pará - Campus Universitário de Santarém. Laboratório de Genética. Santarém, PA, Brasil.Ludwig-Maximilians Universität. Institut für Anthropologie und Humangenetik. Munique, Alemanha.Universidade Federal do Pará. Centro de Ciências Biológicas. Departamento de Genética. Belém, PA, Brasil.Ludwig-Maximilians Universität. Institut für Anthropologie und Humangenetik. Munique, Alemanha.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Centro Nacional de Primatas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Centro de Ciências Biológicas. Departamento de Genética. Belém, PA, Brasil.Ludwig-Maximilians Universität. Institut für Anthropologie und Humangenetik. Munique, Alemanha.This paper presents the karyotype of Callicebus donacophilus pallescens for the first time. The analysis included G-, C-, NOR-banding techniques and FISH with chromosome painting probes from Saguinus oedipus and Homo sapiens. The results were compared with the karyotypes of Callicebus moloch donacophilus and C. moloch previously published. These three karyotypes display the same diploid number (2n=50) but diverge about the number of biarmed and acrocentric chromosomes. The acrocentrics 14 and 15 from C. m. donacophilus and C. moloch have undergone an in-tandem fusion originating a large acrocentric (pair 10) in C. d. pallescens. The major submetacentric pair (pair 1) from C. d. donacophilus and C. moloch have undergone fission originating two acrocentric pairs in C. d. pallescens (pairs 15 and 22). Herein was evidence that, in spite of the high interspecific variation among Callicebus, most of the chromosomes remained conserved

Phylogenetic Reconstruction by Cross-Species Chromosome Painting and G-Banding in Four Species of Phyllostomini Tribe (Chiroptera, Phyllostomidae) in the Brazilian Amazon: An Independent Evidence for Monophyly

<div><p>The subfamily Phyllostominae comprises taxa with a variety of feeding strategies. From the cytogenetic point of view, Phyllostominae shows different rates of chromosomal evolution between genera, with <i>Phyllostomus hastatus</i> probably retaining the ancestral karyotype for the subfamily. Since chromosomal rearrangements occur rarely in the genome and have great value as phylogenetic markers and in taxonomic characterization, we analyzed three species: <i>Lophostoma silvicola</i> (LSI), <i>Phyllostomus discolor</i> (PDI) and <i>Tonatia saurophila</i> (TSA), representing the tribe Phyllostomini, collected in the Amazon region, by classic and molecular cytogenetic techniques in order to reconstruct the phylogenetic relationships within this tribe. LSA has a karyotype of 2n=34 and FN=60, PDI has 2n=32 and FN=60 and TSA has 2n=16 and FN=20. Comparative analysis using G-banding and chromosome painting show that the karyotypic complement of TSA is highly rearranged relative to LSI and PHA, while LSI, PHA and PDI have similar karyotypes, differing by only three chromosome pairs. Nearly all chromosomes of PDI and PHA were conserved <i>in toto</i>, except for chromosome 15 that was changed by a pericentric inversion. A strongly supported phylogeny (bootstrap=100 and Bremer=10 steps), confirms the monophyly of Phyllostomini. In agreement with molecular topologies, TSA was in the basal position, while PHA and LSI formed sister taxa. A few ancestral syntenies are conserved without rearrangements and most associations are autapomorphic traits for <i>Tonatia</i> or plesiomorphic for the three genera analyzed here. The karyotype of TSA is highly derived in relation to that of other phyllostomid bats, differing from the supposed ancestral karyotype of Phyllostomidae by multiple rearrangements. Phylogenies based on chromosomal data are independent evidence for the monophyly of tribe Phyllostomini as determined by molecular topologies and provide additional support for the paraphyly of the genus <i>Tonatia</i> by the exclusion of the genus <i>Lophostoma</i>.</p></div

Cladogram obtained after the analysis by PAUP of the species PHA, PDI, LSI and TSA, using DEC, DYO, and DRO as outgroup using the chromosomal rearrangements as the unique caracters.

<p>Bold numbers indicate Bremer test (above) and bootstrap (below) values for each branch. The numbers refers to the chromosomal changes listed on the Basic Data Matrix (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.s001" target="_blank">S1 Table</a>). All the chromosomal changes (55) were mapped <i>a posteriori</i>, including 32 autapomorphies, 15 synapomorphies, 7 plesiomorphies and one homoplasy.</p

Partial reconstruction of chromosomal changes in phyllostomid bats based on Baker et al.

<p>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.ref015" target="_blank">15</a>] and Hoffmann et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.ref005" target="_blank">5</a>] phylogenies for subfamily Phyllostominae. The chromosome nomenclature followed Pieczarka et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.ref039" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.ref041" target="_blank">41</a>], and refers to homology with PHA chromosomes. Chromosome data for MHI are from Ribas et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.ref036" target="_blank">36</a>], for Desmodontinae are from Sotero-Caio et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.ref040" target="_blank">40</a>], and for PHA and CBR are from Pieczarka et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122845#pone.0122845.ref039" target="_blank">39</a>].</p

Comparative analysis using G-banded chromosomes of PHA, PDI and LSI.

<p>H = constitutive heterochromatin; NOR = Nucleolar Organizer Regions; *Pericentric inversion of pair 15 of PDI to PHA and LSI. Numbers on left: chromosomes from <i>Carollia brevicauda</i>. Numbers below: chromosomes from <i>Phyllostomus hastatus</i>.</p

<i>Tonatia saurophila</i> G-banded karyotype showing homology to <i>Carollia brevicauda</i> (left) and <i>Phyllostomus hastatus</i> (right) chromosomes.

<p><i>Tonatia saurophila</i> G-banded karyotype showing homology to <i>Carollia brevicauda</i> (left) and <i>Phyllostomus hastatus</i> (right) chromosomes.</p

<i>Lophostoma silvicola</i> G-banded karyotype showing homology to CBR (left) and PHA (right) chromosomes.

<p>Box: heterochromatic pairs 15 and Y.</p

Whole chromosome probes FISH of PHA-14 (green) and PHA-15 (red) on PDI chromosomes, showing that PDI chromosomes are homologous to both PHA whole chromosomes their despite of metacentric form.

<p>Whole chromosome probes FISH of PHA-14 (green) and PHA-15 (red) on PDI chromosomes, showing that PDI chromosomes are homologous to both PHA whole chromosomes their despite of metacentric form.</p