Study of the inflammatory activating process in the early stage of Fusobacterium nucleatum infected PDLSCs / 国际口腔科学杂志·英文版

Fusobacterium nucleatum (F. nucleatum) is an early pathogenic colonizer in periodontitis, but the host response to infection with this pathogen remains unclear. In this study, we built an F. nucleatum infectious model with human periodontal ligament stem cells (PDLSCs) and showed that F. nucleatum could inhibit proliferation, and facilitate apoptosis, ferroptosis, and inflammatory cytokine production in a dose-dependent manner. The F. nucleatum adhesin FadA acted as a proinflammatory virulence factor and increased the expression of interleukin(IL)-1β, IL-6 and IL-8. Further study showed that FadA could bind with PEBP1 to activate the Raf1-MAPK and IKK-NF-κB signaling pathways. Time-course RNA-sequencing analyses showed the cascade of gene activation process in PDLSCs with increasing durations of F. nucleatum infection. NFκB1 and NFκB2 upregulated after 3 h of F. nucleatum-infection, and the inflammatory-related genes in the NF-κB signaling pathway were serially elevated with time. Using computational drug repositioning analysis, we predicted and validated that two potential drugs (piperlongumine and fisetin) could attenuate the negative effects of F. nucleatum-infection. Collectively, this study unveils the potential pathogenic mechanisms of F. nucleatum and the host inflammatory response at the early stage of F. nucleatum infection.

Liver cell therapies: cellular sources and grafting strategies / 医学前沿

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver's cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

FOXP4 promotes proliferation of human spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro / 南方医科大学学报

OBJECTIVE@#To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition.@*METHODS@#SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture.@*RESULTS@#The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05).@*CONCLUSION@#In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.

circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis / 国际口腔科学杂志·英文版

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue regeneration. Circular RNAs (circRNAs) have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis. However, how circRNAs regulate hASCs in osteogenesis is still unclear. Herein, we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs. Knockdown of circ_0003204 by siRNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs. We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p. We predicted and confirmed that miR-370-3p had targets in the 3'-UTR of HDAC4 mRNA. The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis. Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model, while overexpression of circ_0003204 inhibited bone defect repair. Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

Roles of ten eleven translocation proteins family and 5-hydroxymethylcytosine in epigenetic regulation of stem cells and regenerative medicine / 北京大学学报(医学版)

The methylation of cytosine is one of the most fundamental epigenetic modifications in mammalian genomes, and is involved in multiple crucial processes including gene expression, cell differentiation, embryo development and oncogenesis. In the past, DNA methylation was thought to be an irreversible process, which could only be diluted passively through DNA replication. It is now becoming increa-singly obvious that DNA demethylation can be an active process and plays a crucial role in biological processes. Ten eleven translocation (TET) proteins are the key factors modulating DNA demethylation. This family contains three members: TET1, TET2 and TET3. Although three TET proteins have relatively conserved catalytic domains, their roles in organisms are not repeated, and their expression has significant cell/organ specificity. TET1 is mainly expressed in embryonic stem cells, TET2 is mainly expressed in hematopoietic system, and TET3 is widely expressed in cerebellum, cortex and hippocampus. This family catalyzes 5-methylcytosine to 5-hydroxymethylcytosine and other oxidative products, reactivates silenced-gene expression, in turn maintains stem cell pluripotency and regulates lineage specification. With the development of tissue engineering, organ transplantation, autologous tissue transplantation and artificial prosthesis have been widely used in clinical treatment, but these technologies have limitations. Regenerative medicine, which uses stem cells and stem cell related factors for treatment, may provide alternative therapeutic strategies for multiple diseases. Among all kinds of human stem cells, adipose-derived stem cells (ADSCs) are the most prospective stem cell lineage since they have no ethical issues and can be easily obtained with large quantities. To date, ADSCs have been shown to have strong proli-feration capacity, secrete numerous soluble factors and have multipotent differentiation ability. However, the underlying mechanism of the proliferation, secretion, acquired pluripotency, and lineage specific differentiation of ADSCs are still largely unknown. Some studies have explored the role of epigenetic regulation and TET protein in embryonic stem cells, but little is known about its role in ADSCs. By studying the roles of TET proteins and 5-hydroxymethylcytosine in ADSCs, we could provide new theoretical foundation for the clinical application of ADSCs and the stem cell-based therapy. In the future, combined with bioprinting technology, ADSCs may be used in tissue and organ regeneration, plastic surgery reconstruction and other broader fields.

Upregulation of IL-10 expression inhibits the proliferation of human periodontal ligament stem cells

Abstract: The abnormal increase in proliferation rate of human periodontal ligament stem cells (PDLSCs) is considered to be involved in the pathogenesis of periodontitis, a disease in which the IL-10-mediated anti-inflammatory pathway plays a critical role. This study aimed to investigate the involvement of microRNA-466l in periodontitis and to explore the possible interaction between IL-10 and microRNA-466l. PDLSCs were obtained from periodontitis-affected teeth and healthy control teeth. The expression of microRNA-466l and IL-10 mRNA was measured in PDLSCs using RT-qPCR. The proliferation ability of PDLSCs was analyzed using CCK-8 assays. Overexpression of microRNA-466l in a PDLSC cell line was established using two different types of PDLSCs, and the effect of microRNA-466l overexpression on IL-10 expression and cell proliferation were detected by western blot and CCK-8 assays, respectively. We found that expression levels of microRNA-466l and IL-10 mRNA were significantly lower (P < 0.05) in PDLSCs derived from periodontitis-affected teeth compared to those derived from healthy teeth. However, the cell proliferation ability was significantly higher in the PDLSCs derived from periodontitis-affected teeth. Meanwhile microRNA-466l overexpression decreased cell proliferation rates of both types of PDLSCs and upregulated IL-10 expression. Together, these data suggest that microRNA-466l can upregulate IL-10 and reduce the proliferation rate of PDLSCs.

Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.

Characterization of victims of aggression and transportation accidents treated at the Forensic Medicine and Dentistry Institute - Campina Grande, Paraíba, Brazil - 2010 / Caracterização de vítimas de agressão e de acidentes de transporte atendidas no Instituto de Medicina e Odontologia Forense - Campina Grande, Paraíba, Brasil - 2010

The objective of this cross-sectional census study was to characterize agression and land-based transport accidents in a city in the Northeast of Brazil. Data was analyzed from live victims who were treated at a forensic service (N = 2.379). In the descriptive analysis, the majority of events were represented by aggression (71.6%); which occurred on weekdays (65%), with 35.1% at night. Trauma occurred to the whole body (63.6%) and to soft tissue (74.2%). On the basis of multiple correspondence analysis, two dimensions were formed: the first dimension (internal reliability = 0.654) was formed by the cause of the event, the trauma and the age group and the second dimension (reliability = 0.514), by age group, occupation and civil status. Three groups with distinct profiles were formed for accidents and aggression: young women who suffered aggression, with trauma to the face and soft tissues during the evening and at weekends; adult men who suffered car accidents, in the morning and on work days; and retired elderly widowers, who were run over.

O objetivo deste estudo tranversal censitário foi caracterizar a agressão e os acidentes de transporte terrestre em uma cidade do nordeste do Brasil. Foram analisados os dados de vítimas vivas que foram atendidas em um serviço forense (N = 2.379). Na análise descritiva, a maioria dos eventos foi a agressão (71,6%); que ocorreu nos dias úteis (65%), sendo 35,1% no período noturno. Os traumas ocorreram no corpo todo (63,6%) e envolveram o tecido mole (74,2%). A partir da análise de correspondência múltipla formaram-se duas dimensões: a primeira dimensão (confiabilidade interna = 0,654) foi formada pela causa do evento, o trauma e a faixa etária e, a segunda dimensão (confiabilidade interna = 0,514), pela faixa etária, a ocupação e o estado civil. Formaram-se três grupos com perfis distintos para os acidentes e agressão. Mulheres jovens que sofreram agressões com traumas faciais, em tecidos moles, no período da tarde e durante os finais de semana. Homens, adultos que sofreram acidentes automobilísticos, pela manhã e em dias úteis, e idosos, viú vos, aposentados e que sofreram atropelamento. Há um elevado número de vítimas de agressão interpessoal, seguido por acidentes de moto e os tipos de acidentes estão associados a grupos populacionais.

Differential expression of stem cell-like proteins in normal, hyperplastic and dysplastic oral epithelium

Objective The identification of stem cells (SC) remains challenging. In the human oral mucosal epithelium, these cells are believed to be in the basal layer (stem cell niche), but their exact location is unclear. The aim of this study was to examine the dysplastic oral epithelium for these SC-like proteins in order to assess their diagnostic value as biomarkers complementing the histological grading of dysplasia. Material and Methods Thirty oral epithelial dysplasia (OED), 25 oral lichen planus (OLP), 10 oral hyperkeratosis and 5 normal oral epithelium (OE) were immunohistochemically examined for four SC markers [integrin β1, neuron-glial-2 (NG2), notch 1 (N1) and keratin 15 (K15)]. Results Three of four SC markers were heterogeneously detected in all samples. K15 overexpression in the lower two-thirds of severe OED suggests an expanded SC niche. Integrin β1 distribution pattern was not measurably different between OEDs and control. NG2 was almost negative to absent in all samples examined. N1 expression was weak and highly variable in normal and dysplastic epithelium, making it an unreliable epithelial stem cell marker. Conclusions Present findings suggest that these markers were unable to identify individual epithelial stem cells. Instead, subpopulations of cells, most probably stem cells and transit amplifying cells with stem cell-like properties were identified in the dysplastic oral epithelium. The characteristic expressions of K15 might be of diagnostic value for oral dysplasia and should be investigated further. .

A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression

PURPOSE: Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects. MATERIALS AND METHODS: In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo. RESULTS: NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons. CONCLUSION: We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.

Aggressive Multilocular Osteoblastoma in the Mandible: A Rare and Difficult Case to Diagnose

Osteoblastoma is a benign neoplasia and is uncommon in the jaws. In some cases, this lesion presents extremely aggressive local characteristics and is termed aggressive osteoblastoma. Because the clinical, radiographic and histopathologic characteristics are similar to those of a variety of benign and malignant tumors, it poses a diagnostic dilemma. This report presents a case of an aggressive osteoblastoma in the mandible and discusses the differential diagnosis of this lesion. A 13-year-old white male sought the Stomatology Clinic at the State University of Paraíba, Campina Grande, PB, Brazil, complaining of asymptomatic swelling on the left side of his face. Cone-beam computerized tomography showed a multilocular, hypodense bone lesion, located in the body of the left mandible and lower third of the ascending ramus. The initial diagnostic hypothesis was juvenile ossifying fibroma or osteosarcoma. After histopathologic examination, the final diagnosis was aggressive osteoblastoma. Surgical resection with a safety margin was performed. There was no evidence of recurrence after a follow-up period of 4 years.

O osteoblastoma é uma neoplasia benigna e incomum nos maxilares. Em alguns casos esta lesão apresenta características locais extremamente agressivas, sendo denominada osteoblastoma agressivo. Devido às características clínicas, radiográficas e histopatológicas serem similares a uma variedade de tumores benignos e malignos, o seu diagnóstico é um dilema. Este relato apresenta o caso de um osteoblastoma agressivo na mandíbula e discute o diagnóstico diferencial desta lesão. Paciente, branco, 13 anos de idade, foi atendido na Clínica de Estomatologia da Universidade Estadual da Paraíba, Campina Grande, PB, Brasil, queixando-se de aumento de volume assintomático do lado esquerdo de sua face. A tomografia computadorizada de feixe cônico revelou uma lesão óssea hipodensa multilocular, localizada no corpo do lado esquerdo da mandíbula e no terço inferior do ramo ascendente da mandíbula. A hipótese diagnóstica foi de fibroma ossificante juvenil e osteosarcoma. Após exame histopatológico, o diagnóstico final foi osteoblastoma agressivo. Foi realizada ressecção cirúrgica com margem de segurança. Não houve sinais de recorrência após 4 anos de acompanhamento.

Utilização de micropartículas biodegradáveis para administração de proteína morfogenética óssea na reconstrução óssea dos maxilares / Use of biodegradable microparticles for administration of bone morphogenetic protein in bone reconstruction of jaws

Atualmente, esforços têm sido concentrados na busca de um implante carreador de proteína morfogenética óssea que possa aperfeiçoar a sua aplicação. As proteínas morfogenéticas ósseas são glicoproteínas responsáveis pelo recrutamento de células osteoprogenitoras para os locais de formação óssea. Dessa maneira, o objetivo deste estudo é revisar a literatura sobre sistemas carreadores para a proteína morfogenética óssea. Estes vêm sendo estudados na tentativa de melhorar o efeito terapêutico através do prolongamento e/ou controle da sua liberação, aumentando, consequentemente, a sua eficácia terapêutica. Essa revisão bibliográfica sugere que as micropartículas biodegradáveis podem ser utilizadas como sistemas carreadores de proteína morfogenética óssea.

Currently, efforts have been concentrated in the search for an implant carrier of bone morphogenetic protein that can enhance its application. The bone morphogenetic proteins are glycoproteins responsible for the recruitment of osteoprogenitor cells to sites of bone formation. Then, the objective of this study was to review the literature about the systems carriers for bone morphogenetic protein. These carriers have been studied in an attempt to improve the therapeutic efficacy by extension and/or control of their release, and therefore increasing their therapeutic efficacy. This literature review suggests that the microparticles can be used as carrier systems for bone morphogenetic protein

Presença de marcadores de células-tronco em linhagens celulares humanas de câncer de mama: possível valor em diagnóstico, prognóstico e terapía / Analysis of stem cells markers in human breast cancer cell lines

O câncer de mama é a doença maligna que mais acomete as mulheres no mundo. Apesar dos inúmeros tratamentos, o óbito se deve principalmente à doença metastática que pode se desenvolver a partir do tumor primário. Esta progressão tumoral decorre da dificuldade de se estabelecer um prognóstico mais preciso. Atualmente, a teoria de células iniciadoras de tumor vem sendo estudada para tentar explicar a biologia do câncer e descrever novos alvos para prognósticos e terapias. O carcinoma mamário foi o primeiro tumor sólido para o qual foi identificada uma subpopulação celular, definida como CD44+/CD24-, apresentando as características de células iniciadoras tumorais. Embora este fenótipo venha sendo muito utilizado para descrever as células iniciadoras tumorais de mama, muitos trabalhos tem questionado a relevância clínica desses marcadores, enfatizando que outros marcadores devem ser identificados. Assim, o objetivo deste trabalho é analisar e caracterizar marcadores de células-tronco que possam estar relacionados com o grau de malignidade no modelo de câncer de mama. Inicialmente, analisou-se a expressão de 10 marcadores de células-tronco em diferentes linhagens de câncer de mama que apresentam graus crescentes de malignidade. O CD90 foi selecionado devido à alta expressão desse marcador na linhagem mais agressiva Hs578T. Para a caracterização deste marcador, realizou-se ensaios funcionais, através do silenciamento do CD90 na linhagem tumorigênica Hs579T e sua superexpressão na linhagem não-tumorigênica MCF10A. As linhagens celulares geradas foram caracterizadas quanto ao crescimento celular, potencial invasivo e metastático. Foi possível observar que houve uma alteração da morfologia nas linhagens transformadas com o CD90 e, também, um maior tempo de dobramento na linhagem Hs578T-CD90- e um menor na MCF10A-CD90+. Além disso, a linhagem MCF10-CD90+ foi capaz de crescer independentemente de EGF. Através da análise da via EGF, foi possível observar que houve um aumento da expressão da forma fosforilada do receptor e dos fatores Erk, c-Jun, e Jnk na linhagem MCF10A-CD90+ e uma diminuição dos mesmos na linhagem Hs578T-CD90-. A análise da atividade do elemento responsivo do fator de transcrição AP1 comprovou que a via de EGF é funcional na linhagem MCF10-CD90+. Também foram analisados os marcadores de transição epitélio-mesenquimal, verificando-se aumento da expressão dos marcadores mesenquimais na linhagem MCF10A-CD90+ e diminuição na linhagem Hs578T-CD90-. Os ensaios in vitro de invasão mostraram que as células MCF10-CD90+ são capazes de migrar e invadir e as células Hs578T-CD90- apresentam diminuição significativa da habilidade de migração e invasão. Além disso, os ensaios de metástase in vitro e in vivo, mostraram que a superexpressão de CD90 levou à malignização das células MCF10A. Por outro lado, a linhagem Hs578T-CD90- apresentou menor potencial metastático in vitro. Portanto, neste trabalho, pela primeira vez, o CD 90 foi caracterizado funcionalmente como um marcador envolvido na transformação maligna do carcinoma mamário, contribuindo, assim, para melhor entendimento da biologia do câncer de mama e para que se possa desenvolver novas ferramentas de diagnóstico/prognóstico e novos protocolos clínicos e terapêuticos

Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols

Bases moleculares da depleção de glutationa sobre a pontencialidade, diferenciação e envelhecimento de células-tronco de pele / Molecular basis of glutathione depletion upon the potenciality, differentiation potential and aging of skin stem cells

A pele está em contínua auto-renovação graças a vários nichos de células-tronco presentes neste tecido. Células progenitoras epidérmicas surgem durante o desenvolvimento embrionário e contribuem para a reposição celular da epiderme durante todo o período de vida dos mamíferos. Neste trabalho, buscou-se analisar o papel da depleção de glutationa durante a estratificação da epiderme embrionária e na manutenção da homeostase no tecido adulto. Encontramos evidências de que este tiol tem um importante papel durante a proliferação da epiderme e formação do folículo capilar. As alterações observadas na ausência de GSH foram relacionados com um padrão diferencial de fosforilação dos fatores de transcrição forkhead-homeobox- tipo-O (FOXO). Em resumo, foi estabelecida uma correlação entre o estado de GSH, a fosforilação de FOXO e o desenvolvimento da epiderme. Para melhor estudar a importância do balanço de GSH, na pele do adulto, e seu papel na manutenção deste tecido, camundongos foram tratados com um inibidor da síntese de GSH e, com N-aceti-lcisteína. Foi observado um aumento da fosforilação de Akt, padrões alterados de fosforilação FOXO e aumento da expressão de genes de genes relacionados à diferenciação. Estes resultados mostram que a via Akt/FOXO desempenha um papel importante na manutenção e diferenciação de células-tronco epidermais. O envelhecimento cronológico leva a alterações morfológicas/funcionais que conduzem à diminuição da auto-renovação, o que ocorre concomitantemente com uma diminuição dos níveis de GSH na pele. Utilizamos, também, animais idosos e avaliamos quais mecanismos eram compartilhados pelo envelhecimento e a depleção deste tiol. Observou-se que uma resposta hiperproliferativa ligada à exaustão de células-tronco pode ser o elo entre a depleção de GSH e o envelhecimento. A influência desse processo também foi investigada no compartimento dérmico, através da análise do impacto da depleção de glutationa sobre a osteogênese de células-tronco mesenquimais murinas. Quando induzidas a se diferenciarem em osso (Alizarin-Red+/Von-Kossa-stain +, aumento dos níveis de mRNA para fosfatase alcalina/osteopontina/osterix), o balanço GSH/GSSG e seu sistema antioxidante correlato é diferencialmente regulado em células-tronco mesenquimais derivadas da derme. Sendo regulado de uma forma redox-dependente através da via de MAPKs. A depleção de GSH leva à diminuição nos níveis de osteogênese em favor da adipogênese, levando ao processo comumente associado ao envelhecimento denominado "adipogenic switc". Em conclusão, os dados obtidos permitem propor um papel central para a glutationa na manutenção/comprometimento de células-tronco na pele

The skin is continuously self-renewing thanks to several stem cell niches. Epidermal progenitor cells arise during embryonic development and contribute to the replenishment of the epidermis during the lifetime of mammals. We set out to analyze the glutathione (GSH) antioxidant system during embryonic epidermis stratification and follicle development and the effect of glutathione withdrawal in this process. We found that glutathione plays an important role during epidermis proliferation and hairshaft formation. The changes observed in the absence of GSH were related to a differential phosphorylation pattern of the forkhead-homeobox-type-O (FOXO) transcription factors. In brief, a correlation between GSH status, FOXO phosphorylation and skin development was established. To further study the importance of GSH in adult skin maintenance and understand the effects of ROS in the Akt/FOXO pathway, we treated cells and mice with an inhibitor of GSH synthesis, and with N-acetyl-cysteine. Increased Akt phosphorylation, altered FOXO phosphorylation patterns and increased gene expression of differentiation-related genes were observed. Our results show that the Akt/FOXO pathway plays an important role in maintenance/differentiation of epidermal stem cells. Chronological ageing leads to morphological/functional changes causing a decline in self-renewal, as well as decreased levels of GSH. We also observed that a cell cycle hyperproliferative response was the link between stem cell exaustion in GSH-depletion and ageing. Dermal mesenchymal stem cells (MSCs), are capable of adipo-chondro- and osteogenesis. Little is known about the impact of ROS in MSC differentiation. We induced murine skin MSCs to differentiate into bone (Alizarin-Red/Von-Kossastain+, increased levels of mRNA for alkalinephosphatase/ osteopontin/osterix). In brief, the balance of GSH/GSSG and related antioxidant system is differentially regulated during this process, found to be regulated in a redox-dependent fashion through the MAPK pathway. When depleted, GSH leads to an adipogenic switch in MSC differentiation. In conclusion, our data leads us to propose a central role for glutathione in the maintenance/commitment of stem cells in skin

Análise in vitro da proliferação, diferenciação e migração de células-tronco de polpa dentária humana em resposta a materiais com potencial para serem empregados como capeadores pulpares diretos (óleo-resina de copaíba isolado ou associado a hidróxido de cálcio ou a agregado de trióxido mineral) / In vitro analysis of proliferation, differentiation and migration of human dental pulp stem cells in response to potential materials for direct pulp capping (oil-resin copaiba alone or associated to calcium hydroxyde or mineral trioxide aggregate)

O hidróxido de cálcio (HCa), o agregado de trióxido mineral (MTA) e o óleo-resina de copaíba (COP) isoladamente apresentam características biológicas do material de capeamento pulpar direto mais apropriado. Com o pressuposto que associados poderiam originar materiais mais apropriados para serem aplicados em capeamento pulpar direto, este estudo objetivou analisar in vitro proliferação, diferenciação e migração de células-tronco de polpa de dente decíduo humano esfoliado (SHEDs) em resposta a substâncias liberadas pelo COP isolado ou associado ao HCa ou ao MTA. Proliferação, diferenciação e migração de SHEDs (Linhagem PDH3) foram analisadas através do ensaio de redução do MTT; da atividade de fosfatase alcalina (ALP), formação de nódulos mineralizados pelo ensaio de Vermelho de Alizarina e expressão dos genes (BGLAP, DSPP, DMP1 e HSP-27) pelo qRT-PCR; e, do ensaio do Scratch, respectivamente. As células foram submetidas à ação de meios condicionados pelos biomateriais, de acordo com os seguintes grupos experimentais: COP isolado (COP); HCa isolado (HCa); HCa associado ao COP (HCa+COP); MTA isolado (MTA) e MTA associado ao COP (MTA+COP). Células crescidas em meio de cultura fresco serviram de controle. Os dados foram comparados utilizando ANOVA complementado pelo teste de Tukey (p 0,05). O grupo HCa apresentou número de células viáveis significativamente menor que o dos demais grupos, inclusive o do grupo HCa+COP (p<0,01) em todos os tempos experimentais...

The calcium hydroxide (CaH), the mineral trioxide aggregate (MTA) and the oil-resin copaiba (COP) by themselves have biological characteristics of the ideal direct pulp capping material. With the assumption that when associated they could originate materials more appropriated for direct pulp capping, this study aimed to analyze the in vitro proliferation, differentiation and migration of stem cells from human exfoliated deciduous teeth (SHEDs) in response to substances leached from COP alone or associated with CaH or MTA. Proliferation, differentiation and migration of SHEDs (PDH3 lineage) were analyzed through the MTT reduction assay; alkaline phosphatase (ALP) activity, mineralized nodules formation using the Alizarin Red assay and gene expression (BGLAP, DSPP, DMP1 e HSP-27) using qRT-PCR; and the Scratch assay, respectively. The cells were submitted to the culture medium conditioned by the biomaterials according to the following experimental groups: COP alone (COP); CaH alone (CaH); CaH associated to COP (CaH+COP); MTA alone (MTA) and MTA associated to COP (MTA+COP). Cells grown in fresh culture medium served as control. Data were compared by ANOVA complemented by the Tukey´s test (p 0.05). The CaH group presented number of viable cells significantly smaller (p<0.01) than those of all other experimental groups, including the CaH+COP, during whole experimental time. The ALP activity in 14 days was similar in all experimental groups. In 21 days, the COP group presented the amount of mineralization higher (p<0.01) than those of all other groups. The gene DMP1 was not expressed by the cells in all experimental groups. The COP group presented the smallest expression (p<0.01) of BGLAP, DSPP and HSP-27 genes. The SHEDs of the MTA+COP group presented superexpression of the BGLAP, DSPP and HSP-27 genes, and in the CaH+COP group the cells superexpressed the BGLAP and HSP-27 genes...

Negative-pressure wound therapy induces endothelial progenitor cell mobilization in diabetic patients with foot infection or skin defects

Non healing chronic wounds are difficult to treat in patients with diabetes and can result in severe medical problems for these patients and for society. Negative-pressure wound therapy (NPWT) has been adopted to treat intractable chronic wounds and has been reported to be effective. However, the mechanisms underlying the effects of this treatment have not been elucidated. To assess the vasculogenic effect of NPWT, we evaluated the systemic mobilization of endothelial progenitor cells (EPCs) during NPWT. Twenty-two of 29 consecutive patients who presented at the clinic of Seoul National Universty Hospital between December 2009 and November 2010 who underwent NPWT for diabetic foot infections or skin ulcers were included in this study. Peripheral blood samples were taken before NPWT (pre-NPWT) and 7-14 days after the initiation of NPWT (during-NPWT). Fluorescence-activated cell sorting (FACS) analysis showed that the number of cells in EPC-enriched fractions increased after NPWT, and the numbers of EPC colony forming units (CFUs) significantly increased during NPWT. We believe that NPWT is useful for treating patients with diabetic foot infections and skin ulcers, especially when these conditions are accompanied by peripheral arterial insufficiency. The systemic mobilization of EPCs during NPWT may be a mechanism for healing intractable wounds in diabetic patients with foot infections or skin defects via the formation of increased granulation tissue with numerous small blood vessels.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Dental pulp stem cells express proteins involved in the local invasiveness of odontogenic myxoma

Little is known about the histogenesis of the odontogenic myxoma (OM). Dental pulp stem cells could be candidate precursors of OM because both OM and the dental pulp share the same embryological origin: the dental papilla. For the purpose of comparing OM and stem cells, this study analyzed the expression of two proteins related to OM invasiveness (MMP-2 and hyaluronic acid) in human immature dental pulp stem cells (hIDPSCs). Three lineages of hIDPSCs from deciduous and permanent teeth were used in this study. Immunofluorescence revealed positive reactions for MMP-2 and hyaluronic acid (HA) in all hIDPSCs. MMP-2 appeared as dots throughout the cytoplasm, whereas HA appeared either as diffuse and irregular dots or as short fibrils throughout the cytoplasm and outside the cell bodies. The gene expression profile of each cell lineage was evaluated using RT-PCR analysis, and HA was expressed more intensively than MMP-2. HA expression was similar among the three hIDPSCs lineages, whereas MMP-2 expression was higher in DL-1 than in the other cell lines. The expression of proteins related to OM invasiveness in hIDPSCs could indicate that OM originates from dental pulp stem cells.

Stem cells and androgen receptor in human periapical periodontitis / Células madre y receptor de andrógenos en periodontitis periapical humana

Activation of macrophages in periapical granulomas occurs through the presence of cytokines, endotoxin and other genetic and epigenetic factors, allowing the initiation of inflammation and bone resorption. The present study aims to analyze the presence of CD133 protein (marker of stem cells) and the AR (androgen receptor) protein in biopsies of human odontogenic periapical granuloma. Biopsies from 14 adult male patients with diagnosis of periapical granuloma included in paraffin blocks were processed histologically to obtain 5-um thick sections. Protein presence was detected and analyzed by immunohistochemistry of CD133 and AR. The quantification considered the number of positive cells in 0.17 mm2 random areas under the microscope using a 1000X objective. Both CD133 and AR proteins are expressed abundantly in cells in pathological periapical granulomas tissue. The number of cells expressing CD133 and AR shows a wide variation coefficient, so its variation is a particular feature for each individual. We concluded that in human odontogenic periapical granuloma there are abundant stem cells and cells expressing AR that may be important for the pathogenic inflammatory process.

La activación de los macrófagos en los granulomas periapicales humanos se producen a través de la presencia de citoquinas, endotoxinas y otros factores genéticos y epigenéticos que permiten la iniciación de la inflamación y la reabsorción ósea. El presente estudio pretende analizar la presencia de proteína CD133 (marcador de células madre) y de la proteína RA (receptor de andrógenos) en las biopsias de granulomas periapicales odontogénicos humanos. Las biopsias de 14 pacientes varones adultos con diagnóstico de granuloma periapical fueron incluidos en bloques de parafina y se procesaron histológicamente para obtener secciones de 5 micras de espesor. La presencia de CD133 y RA fueron detectadas y analizadas por inmunohistoquímica. La cuantificación se realizó considerando el número de células positivas en áreas al azar de 0,17mm2, utilizando microscopio con objetivo de 1000X. Ambas proteínas, CD133 y RA se expresan en abundancia en las células del tejido patológico con granuloma periapical. El número de células que expresan CD133 y RA presentan un amplio coeficiente de variación, por lo que su variación es una característica particular de cada individuo. Se concluye que en granuloma periapical odontogénico humano se expresan abundantes células madre y proteínas receptoras de andrógenos, antecedentes que pueden sermuy importantes en la expresión y diagnosis de los procesos patológicos inflamatorios.