Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding

Abstract

Event Abstract Back to Event Driving iPSC-derived progenitor fate using in vitro synthetized kidney extracellular matrices assembled under macromolecular crowding Valentina Magno1, Bella Roßbach2, Krithika Hariharan2, Jens Friedrichs1, Andreas Kurtz2 and Carsten Werner1, 3 1 Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Germany 2 Charité – Universitätsmedizin Berlin, Berlin-Brandenburg Center for Regenerative Therapies, Germany 3 Center for Regenerative Therapies Dresden, Technische Universität Dresden, Germany Introduction: Cell-derived extracellular matrices (ECMs) have shown in the latest years to have broad application in the field of tissue engineering and regenerative medicine. The recent introduction of macromolecular crowding (MMC) for the modulation of in vitro ECM production allowed, furthermore, to better mimic physiological microenvironments[1]. In this work, we aim to explore the effect of MMC on the secretion of ECM by primary mouse mesangial cells and to study the impact of decellularized ECMs produced under standard and MMC conditions on the growth and differentiation of human induced pluripotent stem cell (iPSC)-derived intermediate mesoderm (IM) progenitors[2]. Methods: Covalently attached mesangial cell-derived ECMs were produced on a maleic anhydride co-polymer platform[3]. The ECM secretion was carried out for 8 days either under standard (Std) or MMC culture conditions by supplementing the medium with the synthetic polysaccharide Ficoll. During the ECM deposition phase, cell viability was assessed using a PrestoBlue assay. Cell layers were decellularised using an hypotonic solution and DNA remnants were removed by DNase treatment. The impact of MMC on ECM composition and morphology was investigated by colorimetric assays, immunohistochemistry, atomic force microscopy and scanning electron microscopy. IM progenitors were seeded in parallel on the mesangial cell-derived ECMs as well as on Geltrex and collagen IV as control substrates, with or without the supplementation of the ROCK inhibitor Y-27632. Adhesion and proliferation of IM progenitors were analyzed respectively using a Crystal Violet and a PrestoBlue assay. Furthermore, the expression of relevant kidney markers was examined via real time PCR. Results and Discussion: PrestoBlue assay showed that MMC did not affect the viability of the mesangial cells during ECM deposition. MMC was found to influence the composition, the mechanical properties and the supramolecular assembly of the mesangial cell-derived ECM. IM progenitors showed significantly higher cell viability and adhesion on both types of ECM (Std and MMC) compared to Geltrex and collagen IV. Particularly, in presence of the ROCK inhibitor, a significantly higher adhesion was found on MMC-ECM compared to both Std-ECM and control substrates. Higher proliferation rates were observed on both types of ECM vs. the controls, with the MMC-ECM outperforming Std-ECM. A significant increase in the expression of some mesangial markers and other glomerular markers was found for IM progenitors cultivated on MMC-ECM vs. Std-ECM, with even less mesangial-like characteristics found on the control substrates. Conclusion: Our results indicate that matrix based signals support the differentiation of the IM progenitors toward the mesangial lineage without the addition of soluble cues. In the future, the combination of cell-derived ECM and MMC may be utilized to study kidney progenitor differentiation and to develop bioengineered kidney tissues. A systematic study on different crowder categories and concentrations may also allow to control progenitor differentiation to obtain a reliable cell source for regenerative approaches. This work was supported by EU Marie Curie NephroTools FP7 Initial Training Network (ITN)