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Summary The specification of the hepatic identity during human liver development is strictly controlled by extrinsic signals, yet it is still not clear how cells respond to these exogenous signals by activating secretory cascades, which are extremely relevant, especially in 3D self-organizing systems. Here, we investigate how the proteins secreted by human pluripotent stem cells (hPSCs) in response to developmental exogenous signals affect the progression from endoderm to the hepatic lineage, including their competence to generate nascent hepatic organoids. By using microfluidic confined environment and stable isotope labeling with amino acids in cell culture-coupled mass spectrometry (SILAC-MS) quantitative proteomic analysis, we find high abundancy of extracellular matrix (ECM)-associated proteins. Hepatic progenitor cells either derived in microfluidics or exposed to exogenous ECM stimuli show a significantly higher potential of forming hepatic organoids that can be rapidly expanded for several passages and further differentiated into functional hepatocytes. These results prove an additional control over the efficiency of hepatic organoid formation and differentiation for downstream applications., Graphical Abstract, Highlights • Microfluidic confined environment enhances hepatic differentiation of hPSCs • SILAC-based proteomic analysis reveals high abundance of secreted ECM proteins • ECM deposition and remodeling correlate with cell-ECM receptor overexpression • Either endogenous or exogenous ECM enhances organoid formation and differentiation, Michielin et al. investigate the secretome of human pluripotent stem cells undergoing hepatic differentiation by coupling microfluidics with SILAC proteomic analysis. They reveal a role of soluble ECM protein accumulation and deposition and leverage these insights to efficiently and robustly derive hepatic organoids from hiPSCs.

Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically., Organoid cultures have been developed from multiple tissues, opening new possibilities for regenerative medicine. Here the authors demonstrate the derivation of GMP-compliant hydrogels from decellularized porcine small intestine which support formation and growth of human gastric, liver, pancreatic and small intestinal organoids.

Cell secretome, the complex set of proteins that are secreted by the cells, is a fundamental mechanism of cell-cell communication both in vitro and in vivo. In vivo, the analysis of proteins secreted into body fluids can bring to the identification of biomarkers for important physiopathological conditions. However, due to the complexity of the protein content of body fluids, a better understanding of the secreted proteins by different cell types is highly desirable and can be performed in vitro for dissection. To this aim, microfluidic culture systems could be particularly relevant because of the accumulation of extrinsic endogenous signals at microliter scale, which better preserves the self-regulation occurring in the small interstitial spaces in vivo. In this work, we perform a quantitative study to compare the secretome in microfluidics and in a standard well plate. Human foreskin fibroblasts are used as a case study. This work also represents an important technological advance in terms of feasibility of high-throughput quantitative protein analyses in microfluidics.