Region 8q24 Is a Susceptibility Locus for Nonsyndromic Oral Clefting in Brazil

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate is a relatively common craniofacial defect with multifactorial inheritance. The association of the rs987525 single nucleotide variant, located in a gene desert at 8q24.21 region, has been consistently replicated in European populations. We performed a structured association approach combined with transcriptional analysis of the MYC gene to dissect the role of rs987525 in oral clefting susceptibility in the ethnically admixed Brazilian population. METHODS: We performed the association study conditioned on the individual ancestry proportions in a sample of 563 patients and 336 controls, and in an independent sample of 221 patients and 261 controls. The correlation between rs987525 genotypes and MYC transcriptional levels in orbicularis oris muscle mesenchymal stem cells was also investigated in 42 patients and 4 controls. RESULTS: We found a significant association in the larger sample (p = 0.0016; OR = 1.80 [95% confidence interval {CI}, 1.21-2.69], for heterozygous genotype, and 2.71 [95% CI, 1.47-4.96] for homozygous genotype). We did not find a significant correlation between rs987525 genotypes and MYC transcriptional levels (p = 0.14; r = -0.22, Spearman Correlation). CONCLUSIONS: We present a positive association of rs987525 in the Brazilian population for the first time, and it is likely that the European contribution to our population is driving this association. We also cannot discard a role of rs987515 in MYC regulation, because this locus behaves as an expression quantitative locus of MYC in another tissue. Birth Defects Research (Part A) 94:464-468, 2012. (C) 2012 Wiley Periodicals, Inc.

Molecular mapping of the regenerative niche in a murine model of myocardial infarction

Adult stem cells are distributed through the whole organism, and present a great potential for the therapy of different types of disease. For the design of efficient therapeutic strategies, it is important to have a more detailed understanding of their basic biological characteristics, as well as of the signals produced by damaged tissues and to which they respond. Myocardial infarction (MI), a disease caused by a lack of blood flow supply in the heart, represents the most common cause of morbidity and mortality in the Western world. Stem cell therapy arises as a promising alternative to conventional treatments, which are often ineffective in preventing loss of cardiomyocytes and fibrosis. Cell therapy protocols must take into account the molecular events that occur in the regenerative niche of MI. In the present study, we investigated the expression profile of ten genes coding for chemokines or cytokines in a murine model of MI, aiming at the characterization of the regenerative niche. MI was induced in adult C57BL/6 mice and heart samples were collected after 24 h and 30 days, as well as from control animals, for quantitative RT-PCR. Expression of the chemokine genes CCL2, CCL3, CCL4, CCL7, CXCL2 and CXCL10 was significantly increased 24 h after infarction, returning to baseline levels on day 30. Expression of the CCL8 gene significantly increased only on day 30, whereas gene expression of CXCL12 and CX3CL1 were not significantly increased in either ischemic period. Finally, expression of the IL-6 gene increased 24 h after infarction and was maintained at a significantly higher level than control samples 30 days later. These results contribute to the better knowledge of the regenerative niche in MI, allowing a more efficient selection or genetic manipulation of cells in therapeutic protocols.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.

Isolamento e caracterização de células-tronco mesenquimais de filtros reutilizáveis e descartáveis de medula óssea

OBJETIVO: Comparar as células-tronco mesenquimais humanas obtidas de filtros de coleta reutilizáveis àquelas coletadas em filtros descartáveis e caracterizá-las utilizando os critérios da International Society for Cellular Therapy. MÉTODOS: Foram isoladas células-tronco mesenquimais humanas de kits de coleta de medula óssea reutilizáveis e descartáveis, pela lavagem dos filtros com meio de cultura. As células isoladas foram caracterizadas de acordo com os critérios estabelecidos pela International Society for Cellular Therapy, por meio das técnicas de citometria de fluxo, diferenciação in vitro e citoquímica. RESULTADOS: As amostras foram obtidas de filtro descartável (n=3) e reutilizável (n=3). Todas as amostras obtidas de filtros descartáveis produziram células-tronco mesenquimais, e todas as células-tronco mesenquimais humanas derivadas de medula óssea preencheram os critérios estabelecidos pela International Society for Cellular Therapy. CONCLUSÃO: Este estudo mostrou que as células-tronco mesenquimais também podem ser obtidas de kits de coleta reutilizáveis (que permanecem em uso em vários centros, no mundo inteiro), para serem empregadas em pesquisa como uma fonte alternativa e ética.

Efeitos de diferentes pressões de aspiração do tecido adiposo na obtenção de células-tronco mesenquimais

INTRODUÇÃO: O enxerto de gordura nos últimos anos voltou a ter destaque como aliado dos cirurgiões plásticos no preenchimento de partes moles, no rejuvenescimento facial volumétrico, nos refinamentos de reconstruções mamárias e por ser rica fonte de células-tronco de comportamento mesenquimal (células-tronco adipoderivadas). Considerando que essas células têm importante papel na angiogênese e na diferenciação adipogênica, com impacto direto na sobrevivência dos enxertos de gordura, determinar parâmetros que otimizem a sua obtenção é imperativo. Nesse contexto, o objetivo deste trabalho é avaliar e comparar dois métodos de obtenção do tecido adiposo da região abdominal quanto ao número de células viáveis presentes na fração vásculo-estromal e analisar a expressão de marcadores de superfície. MÉTODO: Foram selecionadas 9 pacientes do sexo feminino submetidas a lipoaspiração. O tecido adiposo foi obtido da região abdominal infraumbilical. Da metade direita foram coletados 20 ml de gordura, empregando-se cânula acoplada a uma seringa, cujo êmbolo foi tracionado de 2 cc em 2 cc, gerando baixas pressões de aspiração (grupo manual). O mesmo processo foi repetido na metade esquerda, entretanto a cânula estava acoplada a um coletor intermediário estéril e esse a uma máquina de vácuo sob pressão negativa constante de 350 mmHg (grupo a vácuo). As amostras foram centrifugadas e a gordura da camada intermediária dos dois grupos foi submetida a contagem celular, estabelecimento de culturas e posterior imunofenotipagem. RESULTADOS: Este estudo demonstrou que, apesar de não haver diferença estatisticamente significativa, a obtenção da gordura da região abdominal empregando-se lipoaspirador com pressão negativa de 350 mmHg proporcionou maior número de células presentes na fração vásculo-estromal quando comparado à obtenção por meio de seringas de 10 ml, com baixas pressões de aspiração. CONCLUSÕES: O emprego de pressão negativa de 350 mmHg é seguro para a obtenção das células-tronco adipoderivadas e o rendimento celular entre os dois grupos não apresentou diferença estatisticamente significativa.

Cytocompatibility of Porous Biphasic Calcium Phosphate Granules With Human Mesenchymal Cells by a Multiparametric Assay

This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (beta-TCP) (60: 40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100 degrees C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/beta-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aims. Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues. Methods. MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (alpha-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive (R) system. After a mean of 18 +/- 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays. Results. Post-thaw cell recovery: 114 +/- 2.90% (mean +/- SEM). Recovery of viable cells: 93.46 +/- 4.41%, 90.17 +/- 4.55% and 81.03 +/- 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 +/- 1.56%, 72.71 +/- 2.12%, 70.20 +/- 2.39% and 63.02 +/- 2.33% (P<0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 +/- 0.17 X, with a doubling time of 38 +/- 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure. Conclusions. A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.

Bone marrow cells of swine: collection and separation

Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Extrinsic Purinergic Regulation of Neural Stem/Progenitor Cells: Implications for CNS Development and Repair

There has been tremendous progress in understanding neural stem cell (NSC) biology, with genetic and cell biological methods identifying sequential gene expression and molecular interactions guiding NSC specification into distinct neuronal and glial populations during development. Data has emerged on the possible exploitation of NSC-based strategies to repair adult diseased brain. However, despite increased information on lineage specific transcription factors, cell-cycle regulators and epigenetic factors involved in the fate and plasticity of NSCs, understanding of extracellular cues driving the behavior of embryonic and adult NSCs is still very limited. Knowledge of factors regulating brain development is crucial in understanding the pathogenetic mechanisms of brain dysfunction. Since injury-activated repair mechanisms in adult brain often recapitulate ontogenetic events, the identification of these players will also reveal novel regenerative strategies. Here, we highlight the purinergic system as a key emerging player in the endogenous control of NSCs. Purinergic signalling molecules (ATP, UTP and adenosine) act with growth factors in regulating the synchronized proliferation, migration, differentiation and death of NSCs during brain and spinal cord development. At early stages of development, transient and time-specific release of ATP is critical for initiating eye formation; once anatomical CNS structures are defined, purinergic molecules participate in calcium-dependent neuron-glia communication controlling NSC behaviour. When development is complete, some purinergic mechanisms are silenced, but can be re-activated in adult brain after injury, suggesting a role in regeneration and self-repair. Targeting the purinergic system to develop new strategies for neurodevelopmental disorders and neurodegenerative diseases will be also discussed.

Adipose Tissue-Derived Stem Cell Treatment Prevents Renal Disease Progression

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 x 10(5) ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 x 10(5) ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4. IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-alpha, KC, RANTES, and IL-1 alpha. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition.

Reprogramming of prostate cancer-associated stromal cells to embryonic stem-like

BACKGROUND CD90+ prostate cancer-associated (CP) stromal cells represent a diseased cell type found only in tumor tissue. They differ from their normal counterpart in gene expression and inductive signaling. Genetic reprogramming by induced pluripotent stem (iPS) cell technology can effectively change adult cells into stem-like cells through wholesale alteration of the gene expression program. This technology might be used to erase the abnormal gene expression of diseased cells. The resultant iPS cells would no longer express the disease phenotype, and behave like stem cells. METHODS CP stromal cells, isolated from tumor tissue of a surgically resected prostate by anti-CD90-mediated sorting and cultured in vitro, were transfected with in vitro packaged lentiviral expression vectors containing stem cell transcription factor genes POU5F1, LIN28, NANOG, and SOX2. RESULTS Alkaline phosphatase-positive iPS cells were obtained in about 3 weeks post-transfection at a frequency of 10-4. Their colony morphology was indistinguishable from that of human embryonic stem (ES) cells. Transcriptome analysis showed a virtually complete match in gene expression between the iPS and ES cells. CONCLUSIONS Genes of CP stromal cells could be fully inactivated by genetic reprogramming. As a consequence, the disease phenotype was cured. Prostate 72:14531463, 2012. (c) 2012 Wiley Periodicals, Inc.