The Confined Environment in Microfluidics Reveals a Hidden Role of Self-Organizing Extracellular-Matrix Protein Network in the Early Stage of hPSCs Hepatic Differentiation

Abstract

The specification of cell identity during organ development is strictly controlled by extrinsic signals that restrict and define distinct cell fates. However, it is still not clear how cells, when exposed to exogenous signals, activate secretory cascades involving morphogens, growth factors and cytokines, extracellular matrix (ECM) deposition and remodeling. Here, we investigated the proteins secreted by cells in response to developmental exogenous signals, during the progression from endoderm to the hepatic lineage. A microfluidics-based approach coupled with SILAC-MS-based quantitative proteomic analysis revealed, among the 244 endogenously secreted proteins detected, high abundancy of ECM-associated proteins. Hepatocyte-like cells derived in microfluidics, where accumulation of cell-secreted proteins is enhanced up to 7-fold, displayed a more mature hepatic transcriptomic signature and 1.5-fold higher ammonia detoxification capacity compared to conventional culture conditions. Moreover, in the microfluidics-confined environment we observed organized deposition of COL1, FN, LAM and COL4, consistent with observations in human fetal liver at 8-15pcw.Then we tested if the exogenous supplementation of soluble ECM proteins identified by microfluidics-based SILAC-MS proteomic analysis, increase the 3D self-organization capacity of hepatic progenitor cells. We found that FN significantly enhances the hepatic organoid formation capacity of hiPSCs differentiated in conventional culture systems. These nascent organoids can be rapidly expanded for several passages and further differentiated to mature hepatocytes with significant increase of major hepatic functions, including ammonia detoxification, ALB and AAT secretion.