RESUMO
ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/ß-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/ß-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized ß-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and ß-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/ß-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/ß-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.
Assuntos
Astrócitos/metabolismo , Diferenciação Celular , Células-Tronco Neurais/metabolismo , Doenças Neurodegenerativas/terapia , Neurogênese/genética , Neurônios/metabolismo , Transplante de Células-Tronco/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Astrócitos/citologia , Proteína Axina/genética , Proteína Axina/metabolismo , Linhagem Celular , Proteínas Desgrenhadas , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Células-Tronco Neurais/citologia , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Transdução de Sinais/genética , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Human neural progenitor cells (hNPCs) form a new prospect for replacement therapies in the context of neurodegenerative diseases. The Wnt/ß-catenin signaling pathway is known to be involved in the differentiation process of hNPCs. RVM cells form a common cell model of hNPCs for in vitro investigation. Previous observations in RVM cells raise the question of whether observed kinetics of the Wnt/ß-catenin pathway in later differentiation phases are subject to self-induced signaling. However, a concern when investigating RVM cells is that experimental results are possibly biased by the asynchrony of cells w.r.t. the cell cycle. In this paper, we present, based on experimental data, a computational modeling study on the Wnt/ß-catenin signaling pathway in RVM cell populations asynchronously distributed w.r.t. to their cell cycle phases. Therefore, we derive a stochastic model of the pathway in single cells from the reference model in literature and extend it by means of cell populations and cell cycle asynchrony. Based on this, we show that the impact of the cell cycle asynchrony on wet-lab results that average over cell populations is negligible. We then further extend our model and the thus-obtained simulation results provide additional evidence that self-induced Wnt signaling occurs in RVM cells. We further report on significant stochastic effects that directly result from model parameters provided in literature and contradict experimental observations.