RESUMO
Birth defects are the leading cause of infant mortality in the USA, yet the causes of most of these conditions are unknown. While a combination of genetic and environmental factors are suspected in most cases, little information exists about the health risks that prenatal exposure to many common chemicals poses for the fetus. Thus, development and refinement of procedures that can accurately predict embryotoxicity of compounds is important for curtailing the number of infants born with birth defects. The embryonic stem cell test (EST) is a procedure that utilizes comparison of cytotoxicity in embryonic and adult cells and inhibition of differentiation to predict embryotoxicity of compounds tested. Because of its use of existing cell lines, the EST dramatically reduces the need for animal test subjects in toxicity testing. In addition, because of its use of inhibition of differentiation as an endpoint, the EST is extremely versatile in the range of complications it can test for. In this chapter, procedures for use of the validated embryonic stem cell test with the traditional cardiomyocyte differentiation endpoint are explained. The protocol includes discussion of routine stem cell culture, the cardiomyocyte differentiation procedure, and methods for utilization of molecular endpoints for assessing embryotoxicity of compounds.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Murinas/patologia , Miócitos Cardíacos/patologia , Teratogênicos/toxicidade , Testes de Toxicidade/métodos , Animais , Células 3T3 BALB , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacosRESUMO
Embryonic stem cells (ESCs), which are derived from a peri-implantation embryo, are routinely cultured in medium containing diabetic glucose (Glc) concentrations. While pregnancy in women with pre-existing diabetes may result in small embryos, whether such high Glc levels affect ESC growth remains uncovered. We show here that long-term exposure of ESCs to diabetic Glc inhibits their proliferation, thereby mimicking in vivo findings. Molecularly, Glc exposure increased oxidative stress and activated Forkhead box O3a (FOXO3a), promoting increased expression and activity of the ROS-removal enzymes superoxide dismutase and catalase and the cell-cycle inhibitors p21(cip1) and p27(kip1). Diabetic Glc also promoted ß-catenin nuclear localization and the formation of a complex with FOXO3a that localized to the promoters of Sod2, p21(cip1), and potentially p27(kip1). Our results demonstrate an adaptive response to increases in oxidative stress induced by diabetic Glc conditions that promote ROS removal, but also result in a decrease in proliferation.
Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Proteína Forkhead Box O3/genética , Glucose/toxicidade , Estresse Oxidativo/efeitos dos fármacos , beta Catenina/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/toxicidade , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27/genética , Células-Tronco Embrionárias/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Superóxido Dismutase/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
As the worldwide population grows and life expectancies continue to increase, degenerative diseases of the bones, muscles, and connective tissue are a growing problem for society. Current therapies for osteodegenerative disorders such as hormone replacement therapies, calcium/vitamin D supplements and oral bisphosphonates are often inadequate to stop degeneration and/or have serious negative side effects. Thus, there is an urgent need in the medical community for more effective and safer treatments. Stem cell therapies for osteodegenerative disorders have been rigorously explored over the last decade and are yielding some promising results in animal models and clinical trials. Although much work still needs to be done to ensure the safety and efficacy of these therapies, stem cells represent a new frontier of exciting possibilities for bone and cartilage regeneration.