Suppression of the imprinted gene NNAT and X-Chromosome gene activation in isogenic human iPS cells

Genetic comparison between human embryonic stem cells and induced pluripotent stem cells has been hampered by genetic variation. To solve this problem, we have developed an isogenic system that allows direct comparison of induced pluripotent stem cells (hiPSCs) to their genetically matched human embryonic stem cells (hESCs). We show that hiPSCs have a highly similar transcriptome to hESCs. Global transcriptional profiling identified 102-154 genes (\u3e2 fold) that showed a difference between isogenic hiPSCs and hESCs. A stringent analysis identified NNAT as a key imprinted gene that was dysregulated in hiPSCs. Furthermore, a disproportionate number of X-chromosome localized genes were over-expressed in female hiPSCs. Our results indicate that despite a remarkably close transcriptome to hESCs, isogenic hiPSCs have alterations in imprinting and regulation of X-chromosome genes. © 2011 Teichroeb et al

A Dipolar Coupling Based Strategy for Simultaneous Resonance Assignment and Structure Determination of Protein Backbones

A new approach for simultaneous protein backbone resonance assignment and structure determination by NMR is introduced. This approach relies on recent advances in high-resolution NMR spectroscopy that allow observation of anisotropic interactions, such as dipolar couplings, from proteins partially aligned in field ordered media. Residual dipolar couplings are used for both geometric information and a filter in the assembly of residues in a sequential manner. Experimental data were collected in less than one week on a small redox protein, rubredoxin, that was 15N enriched but not enriched above 1% natural abundance in 13C. Given the acceleration possible with partial 13C enrichment, the protocol described should provide a very rapid route to protein structure determination. This is critical for the structural genomics initiative where protein expression and structural determination in a high-throughput manner will be needed

Pluronic F-127 hydrogel as a promising scaffold for encapsulation of dental-derived mesenchymal stem cells.

Dental-derived mesenchymal stem cells (MSCs) provide an advantageous therapeutic option for tissue engineering due to their high accessibility and bioavailability. However, delivering MSCs to defect sites while maintaining a high MSC survival rate is still a critical challenge in MSC-mediated tissue regeneration. Here, we tested the osteogenic and adipogenic differentiation capacity of dental pulp stem cells (DPSCs) in a thermoreversible Pluronic F127 hydrogel scaffold encapsulation system in vitro. DPSCs were encapsulated in Pluronic (®) F-127 hydrogel and stem cell viability, proliferation and differentiation into adipogenic and osteogenic tissues were evaluated. The degradation profile and swelling kinetics of the hydrogel were also analyzed. Our results confirmed that Pluronic F-127 is a promising and non-toxic scaffold for encapsulation of DPSCs as well as control human bone marrow MSCs (hBMMSCs), yielding high stem cell viability and proliferation. Moreover, after 2 weeks of differentiation in vitro, DPSCs as well as hBMMSCs exhibited high levels of mRNA expression for osteogenic and adipogenic gene markers via PCR analysis. Our histochemical staining further confirmed the ability of Pluronic F-127 to direct the differentiation of these stem cells into osteogenic and adipogenic tissues. Furthermore, our results revealed that Pluronic F-127 has a dense tubular and reticular network morphology, which contributes to its high permeability and solubility, consistent with its high degradability in the tested conditions. Altogether, our findings demonstrate that Pluronic F-127 is a promising scaffold for encapsulation of DPSCs and can be considered for cell delivery purposes in tissue engineering

Estresse, estressores e estratégias de enfrentamento de familiares de mulheres acometidas por câncer de mama

O adoecimento de um membro da família provoca alterações na sua dinâmica, e frente ao diagnóstico e ao tratamento do câncer de mama, ela enfrenta uma seqüência de tensões que interferem na unidade familiar. Visando compreender esse processo, realizou-se este estudo que teve como objetivos: caracterizar o familiar de mulheres acometidas por câncer de mama; avaliar os sintomas de estresse, segundo o nível, a freqüência e a intensidade; relacionar o nível de estresse do familiar com as variáveis da paciente (idade, estadiamento, tipo de tratamento e tempo de tratamento) e do próprio familiar (idade, sexo, cor/raça, estado civil, anos de estudo, procedência, religião, ocupação, grau de parentesco e experiência anterior de um membro acometido por câncer); descrever os estressores e identificar as estratégias de enfrentamento desses familiares. Trata-se de um estudo exploratório e descritivo, desenvolvido no Ambulatório Ilza Bianco - Hospital Santa Rita de Cássia, em especial no PREMMA, em Vitória-ES, de agosto a dezembro de 2007, com 200 familiares de mulheres acometidas por câncer de mama. Os instrumentos usados foram Inventário de caracterização da paciente e do familiar, Lista de Sintomas de Stress (LSS/VAS), Descrição dos Estressores e Inventário de Estratégias de Coping de Lazarus e Folkman. Para os dados quantitativos foi realizada a análise descritiva, através de cálculos percentuais, mediana, média e desvio-padrão, com o auxílio do programa SPSS versão 11.0, e aplicação dos testes Qui-quadrado e teste de Fisher para a verificação de relações estatísticas entre as variáveis da mulher, do familiar e o nível de estresse. Para análise dos dados qualitativos, foi utilizado a metodologia de Análise do Discurso do Sujeito Coletivo (DSC). Os resultados demonstraram que: as características dos familiares se assemelham ao perfil da clientela do serviço público de saúde; o nível de estresse foi médio/alto; e que houve uma relação, estatisticamente, significativa entre o nível de estresse e o tempo de tratamento da paciente, a raça/cor e experiência anterior do familiar. Os sintomas mais freqüentes e intensos foram encontrados na esfera cognitiva, comportamental e social e menos freqüentes e intensos, na esfera fisiológica. Os estressores foram a notícia do câncer de mama, o câncer como sentença de morte, o momento da cirurgia, os efeitos adversos da medicação quimioterápica, a incerteza do resultado do tratamento/cura, a convivência com o adoecimento do outro, a solidão do acompanhar e as condições de atendimento do serviço de saúde. As estratégias de enfrentamento mais utilizadas pelos familiares são as centradas no problema, e as menos utilizadas são as focadas na emoção, mostrando que eles estão em busca do re-equilíbrio. Concluiu-se que frente às dificuldades vivenciadas pela família diante do câncer, enfatiza-se a importância do envolvimento da família no processo de tratamento. O profissional de saúde tem o importante papel de compreender, e contextualizar a experiência de cada familiar e paciente e ajudá-los a reconhecer estratégias que amenizem o estresse e os estressores.When a family member gets a disease, it provokes alterations in its dynamics, and front to the diagnosis and the treatment of the breast cancer, they face a sequence of tensions that intervene in the familiar unit. To understand this process, this study took place aiming to characterize the women’s with breast cancer families; assess the symptoms of their stress by the level, the frequency and intensity. Then, it was linked the level of stress with the variables of the patient (age, stage level, , type of treatment and length of treatment) and the family (age, sex, colour / race, marital status, educational background, origin, religion, occupation, degree of relationship and previous experience of a member affected by cancer). We also aim to describe the stressors and identify strategies for confronting these families. This is an exploratory-descriptive study, developed in the Ilza Bianco Ambulatory - Hospital Santa Rita de Cássia, in particular PREMMA in Vitoria-ES, between August and December 2007, with 200 women’s with breast cancer relatives.The instruments used were the Patient and Family Characterization Inventory, the List of Symptoms of Stress (LSS/VAS), Description of Stressors, and, Coping Strategies Inventory of Lazarus and Folkman. To find out the quantitative data it was used the descriptive analysis by listing percentage calculations, median, average and standard deviation. With the help of SPSS version 11.0, it was applied the Chi-Square Test and the Fisher Test for verifying statistics relations between the Women and Families´ variables and the level of stress. As for the Qualitative Data Analysis it was used the Collective Subject Speech Method. We draw conclusions from the results showed: the families’ characteristics are similar to the customers´ profile of the public health. The level of stress was medium/high and there was a statistically significant relationship between the level of stress and patient length of treatment as well as the race / colour and previous experience from a relative. The most frequent and intense symptoms were found within the cognitive, behavioural and social spheres and least frequent and intense within the physiological sphere. The stressors were the announcement of the breast cancer to the family, the cancer as a sentence of death, the surgery, the adverse effects of chemotherapy medication, the uncertainty of the treatment outcome / healing, the coexistence with the one’s illness, the loneliness caused by the one’s cancer, and conditions of the National Heath Service. Coping strategies used by family members are more focused on the problem and few strategies used are focused on emotion, showing that they are looking for re-balance their lives. Furthermore, it was concluded front to difficulties the families were being through, that is important to involve them in the treatment process. The health professional has also an important role in order to understand and contextualize the patient and each family member experience, as well as helping them to recognize strategies that decrease stress and stressors

Application of Stem Cells Derived From the Periodontal Ligament or Gingival Tissue Sources for Tendon Tissue Regeneration.

Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue’s very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P\u3c0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-β3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration

Application of Stem Cells Derived From the Periodontal Ligament or Gingival Tissue Sources for Tendon Tissue Regeneration

Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue’s very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (

Hydrogel Elasticity and Microarchitecture Regulate Dental-Derived Mesenchymal Stem Cells -Host Immune System Cross-Talk

The host immune system (T-lymphocytes and their pro-inflammatory cytokines) has been shown to compromise bone regeneration ability of mesenchymal stem cells (MSCs). We have recently shown that hydrogel, used as an encapsulating biomaterial affects the cross-talk among host immune cells and MSCs. However, the role of hydrogel elasticity and porosity in regulation of cross-talk between dental-derived MSCs and immune cells is unclear. In this study, we demonstrate that the modulus of elasticity and porosity of the scaffold influence T-lymphocyte-dental MSC interplay by regulating the penetration of inflammatory T cells and their cytokines. Moreover, we demonstrated that alginate hydrogels with different elasticity and microporous structure can regulate the viability and determine the fate of the encapsulated MSCs through modulation of NF-kB pathway. Our in vivo data show that alginate hydrogels with smaller pores and higher elasticity could prevent pro-inflammatory cytokine-induced MSC apoptosis by down-regulating the Caspase-3- and 8-associated proapoptotic cascades, leading to higher amounts of ectopic bone regeneration. Additionally, dental-derived MSCs encapsulated in hydrogel with higher elasticity exhibited lower expression levels of NF-kB p65 and Cox-2 in vivo. Taken together, our findings demonstrate that the mechanical characteristics and microarchitecture of the microenvironment encapsulating MSCs, in addition to presence of T-lymphocytes and their pro-inflammatory cytokines, affect the fate of encapsulated dental-derived MSCs

Co-Encapsulation of Anti-BMP2 Monoclonal Antibody and Mesenchymal Stem Cells in Alginate Microspheres for Bone Tissue Engineering

Recently, it has been shown that tethered anti-BMP2 monoclonal antibodies (mAbs) can trap BMP ligands and thus provide BMP inductive signals for osteo-differentiation of progenitor cells. The objectives of this study were to: (1) develop a co-delivery system based on murine anti-BMP2 mAb-loaded alginate microspheres encapsulating human bone marrow mesenchymal stem cells (hBMMSCs); and (2) investigate osteogenic differentiation of encapsulated stem cells in alginate microspheres in vitro and in vivo. Alginate microspheres of 1 ± 0.1 mm diameter were fabricated with 2 × 106 hBMMSCs per mL of alginate. Critical-size calvarial defects (5 mm diameter) were created in immune-compromised mice and alginate microspheres preloaded with anti-BMP mAb encapsulating hBMMSCs were transplanted into defect sites. Alginate microspheres pre-loaded with isotype-matched non-specific antibody was used as the negative control. After 8 weeks, micro CT and histologic analysis were used to analyze bone formation. In vitro analysis demonstrated that anti-BMP2 mAbs tethered BMP2 ligands that can activate the BMP receptors on hBMMSCs. The co-delivery system described herein, significantly enhanced hBMMSC-mediated osteogenesis, as confirmed by the presence of BMP signal pathway-activated osteoblast determinants Runx2 and ALP. Our results highlight the importance of engineering the microenvironment for stem cells, and particularly the value of presenting inductive signals for osteo-differentiation of hBMMSCs by tethering BMP ligands using mAbs. This strategy of engineering the microenvironment with captured BMP signals is a promising modality for repair and regeneration of craniofacial, axial and appendicular bone defects