RESUMO
We identified a human embryonic stem cell subline that fails to respond to the differentiation cues needed to obtain endoderm derivatives, differentiating instead into extra-embryonic mesoderm. RNA-sequencing analysis showed that the subline has hyperactivation of the WNT and BMP4 signalling. Modulation of these pathways with small molecules confirmed them as the cause of the differentiation impairment. While activation of WNT and BMP4 in control cells resulted in a loss of endoderm differentiation and induction of extra-embryonic mesoderm markers, inhibition of these pathways in the subline restored its ability to differentiate. Karyotyping and exome sequencing analysis did not identify any changes in the genome that could account for the pathway deregulation. These findings add to the increasing evidence that different responses of stem cell lines to differentiation protocols are based on genetic and epigenetic factors, inherent to the line or acquired during cell culture.
Assuntos
Proteína Morfogenética Óssea 4/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias Humanas/fisiologia , Proteínas Wnt/genética , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Endoderma/citologia , Endoderma/fisiologia , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/fisiologia , Transdução de Sinais/genética , Transcriptoma , Proteínas Wnt/metabolismoRESUMO
Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-ß- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic.
Assuntos
Diferenciação Celular/genética , Cromossomos Humanos Par 20/genética , Células-Tronco Pluripotentes/citologia , Fator de Crescimento Transformador beta/metabolismo , Aberrações Cromossômicas , Cromossomos Humanos Par 20/química , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Amplificação de Genes , Humanos , Placa Neural/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Sensitivity of neural stem cells viability, proliferation and differentiation upon exposure to methylmercury chloride (MeHgCl) was investigated on different types of biofunctional surfaces. Patterns of biodomains created by microprinting/microspotting of poly-l-lysine or extracellular matrix proteins (fibronectin and vitronectin) allowed for non-specific electrostatic or specific, receptor mediated interactions, respectively, between stem cells and the surface. The neural stem cell line HUCB-NSC has been previously shown to be susceptible to MeHgCl in developmentally dependent manner. Here we demonstrated that developmental sensitivity of HUCB-NSC to MeHgCl depends upon the type of adhesive biomolecules and the geometry of biodomains. Proliferation of HUCB-NSC was diminished in time and MeHgCl concentration dependent manner. In addition, the response to MeHgCl was found to be cell-type dependent. Undifferentiated cells were the most sensitive independently of the type of bioactive domain. Significant decrease of GFAP+ cells was detected among cells growing on poly-l-lysine, while on fibronectin and vitronectin, this effect was observed only in the highest (1µM) concentration of MeHgCl. ß-Tubulin III expressing cells were most sensitive on fibronectin domains. In addition, limited bioactive domains to µm in size, as compared to non-patterned larger area of the same adhesive substrate, exerted protective role. Thus, the surface area and type of cell/biofunctional surface interaction exerted significant influence on developmental stage and cell-type specific response of HUCB-NSC to MeHgCl.