RESUMEN
We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.
Asunto(s)
Apoptosis , Comunicación Celular/fisiología , Uniones Comunicantes/fisiología , Lisofosfolípidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Esfingosina/análogos & derivados , Células Madre/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/farmacología , Comunicación Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Colágeno/farmacología , Conexina 43/metabolismo , Conexinas/metabolismo , Medios de Cultivo Condicionados , Combinación de Medicamentos , Embrión de Mamíferos/citología , Uniones Comunicantes/efectos de los fármacos , Ácido Glicirretínico/farmacología , Humanos , Laminina/farmacología , Ratones , Proteoglicanos/farmacología , Esfingosina/farmacología , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.
Asunto(s)
Lisofosfolípidos/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Esfingosina/análogos & derivados , Células Madre/citología , Células Madre/fisiología , Técnicas de Cultivo de Célula , Células Cultivadas , Investigaciones con Embriones , Humanos , Lisofosfolípidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Ácido Lisofosfatídico/efectos de los fármacos , Receptores del Ácido Lisofosfatídico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Esfingosina/farmacología , Esfingosina/fisiologíaRESUMEN
Gap junctions are intercellular channels that allow both chemical and electrical signaling between two adjacent cells. Gap junction intercellular communication has been implicated in the regulation of various cellular processes, including cell migration, cell proliferation, cell differentiation, and cell apoptosis. This study aimed to determine the presence and functionality of gap junctions in human embryonic stem cells (hESCs). Using reverse transcription--polymerase chain reaction and immunocytochemistry, we demonstrate that human ES cells express two gap junction proteins, connexin 43 and connexin 45. Western blot analysis revealed the presence of three phosphorylated forms (nonphosphorylated [NP], P1, and P2) of connexin 43, NP being prominent. Moreover, scrape loading/dye transfer assay indicates that human ES cells are coupled through functional gap junctions that are inhibited by protein kinase C activation and extracellular signal-regulated kinase inhibition.