Stem cells in embryonic skin development

The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle

Advanced Therapy Medicinal Products in type I diabetes mellitus: technological and regulatory challenges

Introduction: Type 1 Diabetes mellitus (T1DM) is an autoimmune disorder which arises from the destruction of insulin-producing pancreatic β-cells. Currently, Brazil’s advanced therapy medicinal products (ATMP), developed for clinical research and therapeutic purposes, take place in the so-called Cellular Technology Centers (CTC), according to the Resolution nº. 9/2011 of the Collegiate Board of Directors (RDC), enacted by the National Health Surveillance Agency (Anvisa). Objective: This study was conducted with the main objective of describing and discussing the development of ATMP for T1DM treatment. Method: A qualitative research, narrative review and critical discussion of the literature were under taken. Results: ATMP promote new therapeutic approaches for Diabetes, holding great potential to restore the patients’ endogenous insulin secretion, improving their life quality, overcoming the chronic complications of Diabetes and reducing the socioeconomic burden. Nowadays, ATMP in T1DM comprise: a) cell therapy; b) gene therapy products; c) tissue engineering and d) ATMPassociated to biopharmaceutical products. Conclusions: Further research should contribute to stimulate public and private organizations to effectively act towards reducing the impact of Diabetes on individuals and the society as a whole. It is essential that Brazilian legislation closely follows the biotechnological developments, supporting the scientific progress and benefiting T1DM patients with modern and cutting-edge therapies.TÍTULO PT: Produtos Medicinais de Terapia Avançada no diabetes mellitus tipo I: desafios tecnológicos e regulatóriosIntrodução: O diabetes mellitus do tipo 1 (T1DM) é uma desordem autoimune que culmina na destruição das células β-pancreáticas produtoras de insulina. Atualmente, Produtos Medicinais de Terapia Avançada (ATMP) são desenvolvidos no Brasil, para fins de pesquisa clínica e terapia celular, pelos chamados Centros de Tecnologia Celular (CTC), de acordo com a Resolução da Diretoria Colegiada nº 9/2011, emitida pela Agência Nacional de Vigilância Sanitária (Anvisa). Objetivos: Esse estudo foi conduzido com o objetivo principal de descrever e discutir o desenvolvimento de produtos medicinais em terapia avançada (ATMP) para o tratamento de T1DM. Método: Esse estudo foi realizado através de pesquisa qualitativa, revisão narrativa e discussão critica. Resultados: Os ATMP promovem abordagens terapêuticas para o diabetes demonstrando grande potencial para restaurar a secreção endógena de insulina dos pacientes, aumentando sua qualidade de vida, superando as complicações crônicas do Diabetes e reduzindo o impacto socioeconômico desta doença. Atualmente, os ATMP para T1DM compreendem: a) terapia celular; b) terapia gênica; c) engenharia tecidual e d) ATMP associados a produtos biofarmacêuticos. Conclusões: Pesquisas adicionais deverão contribuir para mobilizar governos e organizações para ações que promovam, de forma eficiente, a redução do impacto do diabetes nos indivíduos e na sociedade. Para isso, é essencial que a legislação brasileira acompanhe de perto os desenvolvimentos biotecnológicos, dando suporte para o progresso científico e beneficiando pacientes de T1DM com terapias modernas

Evaluating different methods of microarray data normalization

BACKGROUND: With the development of DNA hybridization microarray technologies, nowadays it is possible to simultaneously assess the expression levels of thousands to tens of thousands of genes. Quantitative comparison of microarrays uncovers distinct patterns of gene expression, which define different cellular phenotypes or cellular responses to drugs. Due to technical biases, normalization of the intensity levels is a pre-requisite to performing further statistical analyses. Therefore, choosing a suitable approach for normalization can be critical, deserving judicious consideration. RESULTS: Here, we considered three commonly used normalization approaches, namely: Loess, Splines and Wavelets, and two non-parametric regression methods, which have yet to be used for normalization, namely, the Kernel smoothing and Support Vector Regression. The results obtained were compared using artificial microarray data and benchmark studies. The results indicate that the Support Vector Regression is the most robust to outliers and that Kernel is the worst normalization technique, while no practical differences were observed between Loess, Splines and Wavelets. CONCLUSION: In face of our results, the Support Vector Regression is favored for microarray normalization due to its superiority when compared to the other methods for its robustness in estimating the normalization curve

Multivariate gene expression analysis reveals functional connectivity changes between normal/tumoral prostates

<p>Abstract</p> <p>Background</p> <p>Prostate cancer is a leading cause of death in the male population, therefore, a comprehensive study about the genes and the molecular networks involved in the tumoral prostate process becomes necessary. In order to understand the biological process behind potential biomarkers, we have analyzed a set of 57 cDNA microarrays containing ~25,000 genes.</p> <p>Results</p> <p>Principal Component Analysis (PCA) combined with the Maximum-entropy Linear Discriminant Analysis (MLDA) were applied in order to identify genes with the most discriminative information between normal and tumoral prostatic tissues. Data analysis was carried out using three different approaches, namely: (i) differences in gene expression levels between normal and tumoral conditions from an univariate point of view; (ii) in a multivariate fashion using MLDA; and (iii) with a dependence network approach. Our results show that malignant transformation in the prostatic tissue is more related to functional connectivity changes in their dependence networks than to differential gene expression. The MYLK, KLK2, KLK3, HAN11, LTF, CSRP1 and TGM4 genes presented significant changes in their functional connectivity between normal and tumoral conditions and were also classified as the top seven most informative genes for the prostate cancer genesis process by our discriminant analysis. Moreover, among the identified genes we found classically known biomarkers and genes which are closely related to tumoral prostate, such as KLK3 and KLK2 and several other potential ones.</p> <p>Conclusion</p> <p>We have demonstrated that changes in functional connectivity may be implicit in the biological process which renders some genes more informative to discriminate between normal and tumoral conditions. Using the proposed method, namely, MLDA, in order to analyze the multivariate characteristic of genes, it was possible to capture the changes in dependence networks which are related to cell transformation.</p

Islet transplantation as a clinical tool: present state and future perspectives

O transplante de ilhotas é um procedimento em desenvolvimento, como alternativa para o tratamento do diabetes tipo 1 que está na fronteira entre o experimental e o clínico. É uma terapia celular na qual as células são implantadas em território diferente do fisiológico em que apenas determinado número incerto conseguirá se adaptar. Aperfeiçoar este processo para obter os mesmos resultados que no transplante de pâncreas, representa um desafio para o qual convergem contribuições da biologia celular, da imunologia e das técnicas de laboratório que se entrelaçam de maneira extremamente complexa. Este trabalho revisa a literatura expondo a evolução do procedimento, a sua metodologia atual e os resultados clínicos obtidos. As perspectivas futuras do transplante diante dos recentes avanços também são discutidas.Islet transplant is an innovative treatment for type 1 diabetic patients, which still lies between experimental and approved transplant therapy. Islet cells are seeded in a non-physiological territory where an uncertain fraction will be able to adapt and survive. Thus, the challenge lies in improving the whole procedure, employing the tools of cell biology, immunology and laboratory techniques, in order to reach the results obtained with whole organ transplant. This review describes the procedure, its progress to the present methodology and clinical results obtained. Future perspectives of islet transplantation in the light of recent biotechnological advances are also focused

Método para clonagem, expressão e purificação do polipeptídeo amilóide das ilhotas pancreáticas humanas

Em 15/04/2016: Anuidade de pedido de patente de invenção no prazo ordinárioDepositadaMétodo para clonagem e expressão do polipeptídeo das ilhotas pancreáticas (IAPP) maduro humano sob a forma recombinante, utilizando um sistema de expressão heteróloga em Escherichia coli baseado no promotor Lacl-T7 RNA polimerase, no qual um fragmento de DNA que contém a seqüência que codifica o peptídeo maduro de IAPP é amplificado por transcrição reversa e reação em cadeia da DNA polimerase (RT-PCR) a partir de RNA total derivado de ilhotas pancreáticas humanas, clonado em vetor de expressão como descrito acima e expresso em bactérias, e método de purificação empregando-se as etapas de lise das células, centrifugação do meio e resuspensão do precipitado em presença de agente desnaturante, centrifugação da solução, remoção do material insolúvel, coleta e filtração do sobrenadante clarificado, ligação da proteína a coluna de afinidade contendo íons metálicos imobilizados, clivagem com protease e eluição da rh-lAPP em forma purificada

Post-Transcriptional Control of RNA Expression in Cancer

Approximately 80% of the human genome contains functional DNA, including protein coding genes, non-protein coding regulatory DNA elements and non-coding RNAs (ncRNAs). An altered transcriptional signature is not only a cause, but also a consequence of the characteristics known as the hallmarks of cancer, such as sustained proliferation, replicative immortality, evasion of growth suppression and apoptotic signals, angiogenesis, invasion, metastasis, evasion of immune destruction and metabolic re-wiring. Post-transcriptional events play a major role in determining this signature, which is evidenced by the fact that alternative RNA splicing takes place in more than half of the human genes, and, among protein coding genes, more than 60% contain at least one conserved miRNA-binding site. In this chapter, we will discuss the involvement of post-transcriptional events, such as RNA processing, the action of non-coding RNAs and RNA decay in cancer development, and how their machinery may be used in cancer diagnosis and treatment

New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Abstract\ud \ud \ud \ud Background\ud Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS.\ud \ud \ud \ud Results\ud From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied.\ud \ud \ud \ud Conclusions\ud Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.We would like to thank Marc Sylvester for his invaluable advices regarding quantitative proteomics, Marcella Nunes de Melo Braga (University of Southern Denmark) for helping to set up the dimethyl labeling, Giuseppe Palmisano (Harvard University) and Melanie Schultz (University of Southern Denmark) for advices regarding sample analysis in MS sample preparation and data analysis. The excellent technical assistance of Zizi de Mendonça, Debora Costa Lopes and Marluce C. Mantovani is also acknowledged. This project was supported by: BNDES, CAPES, CNPq, FAPESP (grant number 2008/53974-4),MCTI and MS-DECIT

Analysis of the role of hormones and growth factors in the control of cell proliferation in mammals

O objetivo deste trabalho foi estudar o processo pelo qual hormônios e fatores de crescimento controlam a proliferação celular em mamíferos. O modelo experimental utilizado foi linhagens de células estabelecidas em cultura. Os estudos centraram-se em dois tipos básicos de células: fibroblastos e células adrenais e o ataque experimental foi feito sob dois pontos de vista: bioquímico e genético. O ataque bioquímico envolveu desenvolver estudos cinéticos da síntese de DNA não só durante o carenciamento de células para soro, como também durante a reestimulação de células carenciadas por:soro, hormônios e fatores de crescimento. Medidas do conteúdo intracelular de cAMP foram efetuadas com o intuito de adquirir informações à respeito do mecanismo de ação destes fatores. Um modelo de ciclo celular foi proposto no qual o controle do crescimento seria exercido através de reguladores positivos e negativos que agiriam estimulando ou inibindo a passagem de células do estado de repouso (Go) para a fase proliferativa. Entre os reguladores positivos (estimuladores) do sistema fibroblasto, encontra-se hormônios clássicos, como esteróides e insulina, e fatores de crescimento de natureza hormonal como EGF, PF (fator proteico extraído de glândulas pituitárias) e prostaglandina F2&#945;. O esteróide hidrocortisona pode agir como regulador negativo, inibindo o crescimento de fibroblastos. Medidas do período de tempo transcorrido desde a estimulação de células carenciadas (Go), até o aparecimento da onda de síntese de DNA (período definido operacionalmente como Gl) foram feitas. Em fibroblastos 3T3 este período foi de 12 a 13 horas tanto para células estimuladas com soro como com hormônios clássicos (hidrocortisona, insulina) ou fatores de crescimento (EGF, PF) ou ainda com combinações deles (EGF + PF + insulina; PF + hidrocortisona; PF + hidrocortisona + insulina). No sistema células adrenais, adrenocorticotropina (ACTH) foi o único hormônio clássico que apresentou atividade sobre o crescimento destas células e também o único efetuador negativo encontrado. Neste sistema PF mostrou-se como o único fator com atividade estimulatória sobre o crescimento. Gl aqui foi de 11 horas tanto para células estimuladas com soro como com PF. Além disso os hormônios clássicos hidrocortisona e insulina não apresentaram atividade estimulatória por si só ou em combinação com PF. A análise da ação de hidrocortisona no sistema fibroblasto e de ACTH no sistema células adrenais estimuladas, forneceu evidências de que após deixar Go, em direção a S, numa certa altura de Gl as células tornam-se irreversivelmente comprometidas com o processo replicativo. Este comprometimento parece ocorrer 5 horas antes de S, sendo referido como Glc. Em face destes resultados foi proposto que os reguladores agem estimulando ou inibindo a transição Go &#8594; Glc. Na tentativa de obter maior definição do sistema de controle do crescimento, aproveitamo-nos das vantagens oferecidas pelo modelo experimental usado, para a busca de mutantes do tipo regulatório. Esta busca resultou no isolamento das linhagens ST1 e AR-1, derivadas, respectivamente, de fibroblastos 3T3 e células adrenais Y-l. Entre os vários aspectos interessantes da linhagem ST1 destaca-se: a) o dramático efeito de hidrocortisona causando mudança nas características das células as quais passam de um fenótipo tipicamente transformado para normal. Este fenômeno foi observado tanto \"in vitro\" (através de medidas de parâmetros de crescimento) como \"in vivo\" (através de ensaios de tumorogenicidade); b) as alterações morfológicas de caráter antagônico provocadas, por um lado, pela adição de hidrocortisona (causando achatamento) e, por outro, pela retirada do soro ou adição de cAMP ao meio de cultura (arredondamento). Através do estudo da ação de inibidores, obteve-se evidências do envolvimento de microtúbulos nestas alterações morfológicas. A análise do conteúdo intracelular de cAMP indicou que este nucleotídeo não atua como mediador da ação de hidrocortisona. Sua ação parece ser devida à indução de alterações no sistema superfície celular - membrana -citoesqueleto. Ao contrário de outros variantes de células Y-l resistentes à ACTH, células AR-1 mostraram-se também resistentes a cAMP. A utilidade destas células nos estudos da postulada mediação deste nucleotídeo na ação de ACTH, é óbvia.The aim of this work was to study the process by which hormones and growth factors control proliferation of mammalian cells. Cell lines established in culture were used as the experimental model. The studies were centered on two basic types of cells: fibroblasts and adrenal cells and the experimental approach was made from two viewpoints: biochemical and genetic. The biochemical approach involved kinetic studies of the DNA synthesis process not only during serum starvation but also during restimulation of serum starved cells by serum, hormones and growth factors. Intracellular cyclic AMP determinations were made in order to gain informations on the mechanism of action of these factors. A cell cycle model was proposed in which cell growth control would be exerted by positive and negative regulators that would act by stimulating or inhibiting the flow of cells from a resting state (Go) to the proliferative phase. Among the positive regulators (stimulators) found for the fibroblast system are: classical hormones, like steroids and insulin, and growth factors of homonal nature, like EGF, PF (protein factor extracted from pituitary glands) and prostaglandin F2&#945;. The steroid hydrocortisone can also act as a negative regulator, inhibiting fibroblast growth. Measurements of the time interval between stimulation of serum starved (Go) cells and the onset of DNA synthesis (period that is operationally defined as Gl) were made. In 3T3 fibroblasts this period was 12 to 13 hours for cells stimulated not only by serum but also by classical hormones (hydrocortisone, insulin) or growth factors (EGF, PF) or even by combinations of these factors (EGF + PF + insulin; PF + hydrocortisone; PF + hydrocortisone + insulin). In the adrenal system, adrenocorticotropin (ACTH) was the only classical hormone to present activity on the growth of these cells and also the only negative regulator found. In this system PF was shown to be the only factor with growth stimulatory activity. GL was estimated as 11 hours for cells stimulated with serum or PF. Moreover hydrocortisone and insulin had no stimulatory activity \"per si\" or in combination with PF. The analysis of hydrocortisone action on the fibroblast system on one hand and of that of ACTH on the adrenal system, on the other, indicated that upon leaving Go, towards S, at a certain point in Gl, cells become irreversibly committed to the replicative process. This commitment seems to occur 5 hours before S and is referred to as Glc. In view of these data we proposed that regulators act by stimulating or inhibiting the transition Go &#8594; Glc. In an attempt to obtain a better definition of the growth control system and taking advantage of the experimental model utilized, we searched for mutants of the regulatory type. This search resulted in the isolation of the lines ST1 and AR-l from 3T3 fibroblasts and Y-l adrenal cells, respectively. Among several interesting aspects of the STl cell line, we point out: a) the dramatic effect of hydrocortisone changing the characteristics of these cells from a typically transformed phenotype to a normal pattern. This phenomenon was observed both \"in vitro\" (by measuring a number of growth parameters) and \"in vivo\" (by tumorogenicity assays). b) morphological alterations of antagonistic nature caused by hydrocortisone (flattening) on one hand, and by the removal of serum or cAMP addition to the culture medium (rounding) on the other. Evidence for the involvement of microtubules in these alterations were obtained through studies on the action of several inhibitors. Quantitative analysis of intracellular cAMP indicated that this nucleotide does not act as a mediator of hydrocortisone action. Rather, this action seems to be due to the induction of alterations on the cell surface-membrane-cytoskeleton system. Contrary to other variants of the Y-1 line which are resistant to ACTH, AR-1 cells are also resistant to cAMP. The usefulness of these cells in studies of the postulated mediation by cAMP of the ACTH action, is obvious