RESUMO
BACKGROUND: Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca2+ dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time. METHODS: The 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous and mechanically induced intracellular Ca2+ activity was measured with live-cell Ca2+ imaging either immediately or 7 days after wounding. The healing time and speed were analyzed with time-lapse bright field microscopy. The Ca2+ activity and healing speed were analysed with image analysis. In addition the extracellular matrix deposition was assessed with confocal microscopy. RESULTS: The Ca2+ dynamics in hESC-RPE monolayers differed depending on the culture time: 9-day-cultured cells had higher number of cells with spontaneous Ca2+ activity close to freshly wounded edge compared to control areas, whereas in 28-day-cultured cells there was no difference in wounded and control areas. The 28-day-cultured, wounded and 7-day-healed hESC-RPEs produced wide-spreading intercellular Ca2+ waves upon mechanical stimulation, while in controls propagation was restricted. Most importantly, both wave spreading and spontaneous Ca2+ activity of cells within the healed area, as well as the cell morphology of 28-day-cultured, wounded and thereafter 7-day-healed areas resembled the 9-day-cultured hESC-RPEs. CONCLUSIONS: This acquired knowledge about Ca2+ dynamics of wounded hESC-RPE monolayers is important for understanding the dynamics of RPE wound healing, and could offer a reliable functionality test for RPE cells. The data presented in here suggests that assessment of Ca2+ dynamics analysed with image analysis could be used as a reliable non-invasive functionality test for RPE cells.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Epitélio Pigmentado da Retina/citologia , Cicatrização , Diferenciação Celular , HumanosRESUMO
Ca2+ is a second messenger controlling vital cellular processes, including cell maturation. Changes in Ca2+ signaling during maturation of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have not been assessed previously. The aim of this study was to investigate maturation-dependent changes in transient intracellular Ca2+ ([Ca2+] i ) increases in hESC-RPE. For this, we developed image analysis tools to evaluate cell-specific Ca2+ signals from the entire field of view. Spontaneous and mechanically induced transient [Ca2+] i increases (STIs and MITIs) were analyzed in hESC-RPEs cultured for 9 or 28 days, altogether from more than 80,000 cells. Both cultures showed STIs: the longer culture time resulted in twofold increase of amount of cells with STIs. Mechanical stimulation induced intercellular Ca2+ waves in cells from both time points, but longer culture time reduced Ca2+ wave spreading. Depletion of intracellular Ca2+ stores decreased cell fraction with STIs and MITIs at both time points, and absence of extracellular Ca2+ had similar effect on cells with STIs. To conclude, hESC-RPE cells undergo significant Ca2+ signaling re-arrangements during a short maturation period increasing cell fraction with STIs, while decreasing coordinated cell response to mechanical stimulation. This knowledge and proposed analysis tools can be used for assessment of hESC-RPE maturation in vitro.
Assuntos
Sinalização do Cálcio/fisiologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Linhagem Celular , HumanosRESUMO
OBJECTIVE: Computational models of calcium (Ca²âº) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca²âº signaling model for RPE based on our experimental data of mechanically induced Ca²âº wave in the in vitro model of RPE, the ARPE-19 monolayer. METHODS: We combined six essential Ca²âº signaling components into a model: stretch-sensitive Ca²âº channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca²âº pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca²âº waves reproduced our control experimental data and data where gap junctions were blocked. RESULTS: Our model was able to explain Ca²âº signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca²âº through plasma membrane SSCCs and gap junctions conduct the Ca²âº and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca²âº signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell's sensitivity to the extracellular ligand attenuating the Ca²âº signal in the distance. CONCLUSIONS: The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca²âº wave attenuation by suramin. Being the first RPE Ca²âº signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.