Project description:Background: The main focus of the work was the evaluation of gene expression differences between our established NSCLC 3D cell culture model and the 2D cell culture in regard to the use of our model for drug screening applications. Methods: The non-small cell lung cancer (NSCLC) cell lines Colo699 and A549 were cultivated as monolayer (2D) on cell culture plates for five days or as microtissues (3D) in a hanging-drop system for five and ten days, respectively. Cells and microtissues were harvested and Affymetrix chip analyses were performed with the prior isolated RNA. This was repeated in three independent experiments. Subsequent biostatistical data analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. Results: The analyses revealed a high interassay correlation within the distinct culture systems, thus proving a high validity of our data. The comparison of 3D versus 2D cell cultures revealed significant differences in RNA expression (979 genes for A549; 1106 genes for Colo699), but the overlap of changes in RNA profiles between the cell lines at the individual gene level was small (149 genes), potentially reflecting overall heterogeneity and their origin, i.e. primary vs pleural effusion. Nevertheless, these RNA expression changes affected most relevant cancer-associated pathways as DNA methylation, cell cycle, rRNA expression and meiosis pathways. Furthermore, the expression differences between 2D and 3D were more evident after longer cultivation time, which supports the hypothesis of cultivation related mechanisms and the usage of long-time cultivation systems. Conclusion: In summary, our data support the need of innovative 3D drug testing systems to close the gap between in-vitro drug screening and in-vivo data. Thus, our 3D NSCLC model might provide a model to address the challenge of microenviroment associated resistance mechanisms, as well as cell-cell interaction related effects.

Project description:The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during the stressful events surrounding early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.

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Generation and Maturation of Human iPSC-derived 3D Organotypic Cardiac Microtissues in Long-Term Culture

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