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Poster presented at 2018 EASD meeting concerning the different GIP and GLP-1 mechanisms of action.

Stem cells are undifferentiated cells that have the capability to differentiate into specialized cells and to subdivide indefinitely to produce more stem cells. Studies on human stem cells represent a powerful method to analyze biological and physiological processes specific of human cells, as well as for tissue engineering, regenerative medicine and cell therapies. When these studies are performed in vitro it is important to take in account that cellular processes such as adhesion, migration, growth, secretion, and gene expression are triggered, controlled, or influenced by the biomolecular three-dimensional organization of cell-surface contact. This organization cannot be straightforwardly reproduced in the laboratory; however modern methods in microtechnology enable researchers to generate complex environments at a biologically appropriate resolution. In this work we employ microfabricated and photopatterned polymeric substrates to reproduce a 3D environment morphologically and chemically tuned to induce specific cell responses (from cell adhesion to cell shape) and to detect specific biological parameters relevant for human stem cells both at the pluripotent and at the differentiated stage. In particular, at the pluripotent stage, we focus our attention on the response of the cell nucleus to specific microenvironments of the cells, as nuclear shape and structure are recognized to be deeply related with cell function during developmental, physiological and pathological changes which occur during the life time of the cells. The aim of our investigations is to understand if nuclear morphological reorganization caused by the microenvironment of the cell could affect the further behavior of the cells, especially the gene expression. We employ micropillared substrates where the pillar to pillar distance is smaller than the cell size and in some cases even smaller than the cell nuleus to compare nuclear behavior of human pluripotent stem cells (hPSCs) and differentiated cells, especially to elucidate to what extent the microenvironment influences the shape of the nucleus. Nuclear deformability is quantified by analyzing the shape of fluorescent labeled nuclei on images acquired by fluorescent or confocal microscopy. It is observed that the cell geometry of human pluripotent stem cells seeded on microstructured substrates is strongly altered by through contact with the microstructured substrates. This includes even a strong deformation of the nucleus that penetrates among the microstructures. In contrast to this, differentiated cells show much less deformable nuclei. To obtain information on a possible relationship between nuclear shape and cell function, we observe the nuclear shape and quantify nuclear deformability at the pluripotent stage and along the process of differentiation. In addition, we compare nuclear behavior and gene expression between cells seeded and differentiated on flat and on microstructured substrates. Our results show that pluripotency of hPSCs is maintained regardless whether the cells are seeded on flat or microstructured substrates. In contrast to this, gene expression analysis of endoderm, mesoderm and ectoderm germ layers, reveal that the morphological reorgaization of the nuclei, which is observed for cells seeded on microstructured substrates, can hinder the process of endoderm specification. Ectoderm and mesoderm early germ layer committment take place despite the highly deformed shape of the nuclei seeded on microstructured substrates, however ectoderm specification takes place along with a process of nuclear stiffening. Along ectoderm committment, cell nuclei raise up from micropillar interspaces and assume a round shape on top of the pillared surfaces. Such nuclear stiffening must be related to a structural and functional reorganization of cytoskeletal and nuclear envelope proteins. The use of a simple substrate fabrication approach combined with image analysis, allows to quantify variation in nuclear shape of hPSCs in a straightforward way. Thus, such approach can be combined in the future with a wider parallel analysis of protein expression to associate nuclear shape variation to specific changes in gene expression. The developed platform consisting of microfabrication and image analysis is also used to study a second system. We realize here a platform that allows the quantification of physiological parameters of cells derived by differentiation of hPSCs, to evaluate the functional maturation of the investigated cells. In particular, we focus our attention on cardiomyocytes derived from hPSCs. These cells are of special interest since heart diseases are the leading cause of mortality in the Western Countries. In the last decades much effort was put in the research for new approaches in treatment of infarcted hearts as well as congenital heart diseases, spanning from pharmacological to cell therapies. Both need reliable representative study models at the cellular level, as close as possible to the human physiology. To reach this goal we realize an in vitro set up that allows the simultaneous recording and analysis of calcium dynamics and contraction force of cardiomyocytes which have been directly derived from human pluripotent stem cells (hPSC-CMs). To tune cell response after seeding, we fabricate our substrates so that cell adhesion can be locally controlled. To this, we combine a cell-adhesive photopolymerizable elastomeric polymer, for the generation of a micropillared substrate obtained by replica molding, with a cell-repellent material. The cell-adhesive photoreactive polymer is a copolymer of n-butylacrylate and methacryloyl-4-oxy-benzophenone, P(nBA-co-%MABP). The cell-repellent polymer is polyacrylamide (PAA). To guide cell adhesion and shape, the cell-repellent PAA is attached and crosslinked to the rubber surface by UV light exposure through photolithography. The thus obtained substrate consists of elastomeric micropillars with cell-attractive pillar heads onto which hPSC-CMs selectively grew and aligne assuming an elongated shape. Calcium dynamics is acquired through confocal analysis, where the transient of calcium is detected by intensity changes of the fluorescent probe Fluo-A, which serves as a calcium indicator. Micropillar deflection caused by cellular contraction and relaxation is quantified by analysis of confocal images. The deflection data are converted into (contraction) force values by using a finite elements model. We apply the developed platform to compare calcium transient and contraction force of hPSC-CMs after 1 week and 5 weeks from seeding to study whether or not functional maturation of the cells occurrs in this time gap. This would be a crucial factor for their employment in drug testing and in the early pharmacological development pipeline. We report that hPSC-CMs cultured on our morpologically and chemically tuned substrates enhanced their functional properties from week 1 to week 5 of culture. In particular, the duration of the calcium transients was found to decrease, while the contraction force was found to increase on cells that were kept in culture for B weeks on microstructured and photopatterned substrates. The use of microtechnology techniques to provide controlled microenvironment combined with stem cells, such as hPSCs and hPSC-CMs goes into the direction to realize in vitro biological assays of cells that are sensitive to the mechanical stimuli from the environment. The generation of soft, deformable matrices with local control of the surface chemistry mimics more closely the behavior of regular cells under physiological conditions. As the cells used in a 3D-microenvironment study are actually human cells, it could be possible to limit errors caused by the difference between human and animal physiology. The described approach can reduce the need of more expensive and often not satisfactory in vivo studies, i.e. animal testing, where ethical aspects are under strong debate. Such replacement of animal testing could be used for drug screening and for fundamental studies of the mechanisms triggered in cells in response to topographical, chemical or mechanical modifications of the cell microenvironment

Nuclear deformation is an essential phenomenon allowing cell migration and can be observed in association with pathological conditions such as laminopathies, neurodegenerative disorders and diabetes. Abnormal nuclear morphologies are a hallmark of cancer progression and nuclear deformability is a necessary feature for metastatic progression. Nevertheless, the cellular processes and the key molecular components controlling nuclear shape are poorly understood, in part due to a limited availability of assays that allow high-throughput screening of nuclear morphology-phenotypes. In this study, we explore the application of micropillared substrates as the basis for a phenotypic screening platform aimed at identifying novel determinants of nuclear morphology. We designed PDMS substrates to maximize simplicity in image acquisition and analyses, and in a small-scale screening of inhibitors targeting chromatin-modifying enzymes, we identify histone deacetylation as cellular process involved in nuclear deformation. With increasingly specific targeting approaches, we identify HDAC2 as a novel player in controlling nuclear morphology through gene transcription repression. This study shows the effectiveness of micropillar-based substrates to act as phenotypic drug screening platforms and opens a new avenue in the identification of genes involved in determining the nuclear shape.

Nuclear shape modulates cell behavior and function, while aberrant nuclear morphologies correlate with pathological phenotype severity. Nevertheless, functions of specific nuclear morphological features and underlying molecular mechanisms remain poorly understood. Here, we investigate a nucleus-intrinsic mechanism driving nuclear lobulation and segmentation concurrent with granulocyte specification, independently from extracellular forces and cytosolic cytoskeleton contributions. Transcriptomic regulation of cholesterol biosynthesis is equally concurrent with nuclear remodeling. Its putative role as a regulatory element is supported by morphological aberrations observed upon pharmacological impairment of several enzymatic steps of the pathway, most prominently the sterol ∆14-reductase activity of laminB-receptor and protein prenylation. Thus, we support the hypothesis of a nuclear-intrinsic mechanism for nuclear shape control with the putative involvement of the recently discovered GGTase III complex. Such process could be independent from or complementary to the better studied cytoskeleton-based nuclear remodeling essential for cell migration in both physiological and pathological contexts such as immune system function and cancer metastasis.