RESUMO
The pro-neural transcription factor neurogenin-2 (NGN2) possesses the ability to rapidly and effectively transform stem cells into fully operational neurons. Here we report the successful generation of a modified H9 human embryonic H9 stem cell line containing a doxycycline (DOX) inducible NGN2 expression construct featuring a floxed Blasticidin/mApple selection module in the safe-harbor locus CLYBL. This cell line retains its pluripotent state in the absence of DOX, yet readily transitions into a neuronal state upon DOX introduction.
Assuntos
Doxiciclina , Edição de Genes , Humanos , Doxiciclina/farmacologia , Linhagem Celular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias/metabolismo , Diferenciação Celular/fisiologiaRESUMO
Directed neuronal differentiation of human pluripotent stem cells (hPSCs), neural progenitors, or fibroblasts using transcription factors has allowed for the rapid and highly reproducible differentiation of mature and functional neurons. Exogenous expression of the transcription factor Neurogenin-2 (NGN2) has been widely used to generate different populations of neurons, which have been used in neurodevelopment studies, disease modeling, drug screening, and neuronal replacement therapies. Could NGN2 be a "one-glove-fits-all" approach for neuronal differentiations? This review summarizes the cellular roles of NGN2 and describes the applications and limitations of using NGN2 for the rapid and directed differentiation of neurons.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem da Célula/genética , Terapia Baseada em Transplante de Células e Tecidos , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Cortical neurospheres (NSPs) derived from human pluripotent stem cells (hPSC), have proven to be a successful platform to investigate human brain development and neuro-related diseases. Currently, many of the standard hPSC neural differentiation media, use concentrations of glucose (approximately 17.5-25 mM) and insulin (approximately 3.2 µM) that are much greater than the physiological concentrations found in the human brain. These culture conditions make it difficult to analyse perturbations of glucose or insulin on neuronal development and differentiation. We established a new hPSC neural differentiation medium that incorporated physiological brain concentrations of glucose (2.5 mM) and significantly reduced insulin levels (0.86 µM). This medium supported hPSC neural induction and formation of cortical NSPs. The revised hPSC neural differentiation medium, may provide an improved platform to model brain development and to investigate neural differentiation signalling pathways impacted by abnormal glucose and insulin levels.
