GSE159926
The confined environment in microfluidic reveals a hidden role of self-organizing extracellular-matrix in hepatic commitment and organoid formation of hiPSCs
The specification of cell identity during human liver 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, which are extremely relevant especially in 3D self-organizing system such as hepatic organoids. Here, we investigated 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 a microfluidics-based approach coupled with SILAC-MS-based quantitative proteomic analysis we found high abundancy of ECM-associated proteins. Hepatocyte-like cells derived in microfluidics, where accumulation of cell-secreted proteins is enhanced up to 7-fold, showed organized deposition of COL1, FN, LAM and COL4, consistent with observations in human fetal liver at 8-15pcw. Further differentiation leads to a more mature hepatic transcriptomic signature and 1.5-fold higher ammonia detoxification capacity compared to conventional culture conditions. Hepatic committed cells in an early stage either derived in microfluidics and exposed to exogenous ECM stimuli show a significant higher potential for hepatic organoids formation. Nascent organoids can be rapidly expanded for several passages and further differentiated into functional hepatocytes. These results proved an additional control over the efficiency of hepatic organoid formation and differentiation for downstream applications. Overall design: Comparison of mature hepatocyte cells derived in microfluidics and conventional culture system from hiPSCs. Pluripotent cells were used as undifferentiated control.