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The confined environment in microfluidic reveals a hidden role of self-organizing extracellular-matrix in hepatic commitment and organoid formation of hiPSCs

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE159926 | GEO | 2020/10/24

REPOSITORIES: GEO

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Project description:Cholangiocarcinoma (CCA) is a hepatobiliary malignancy with dismal prognosis. Currently available models fail to recapitulate the full complexity of CCA, particularly the desmoplastic environment and the interplay between cancer cells and the extracellular matrix (ECM). We aimed to study the role of epithelial tumor cells in ECM deposition and desmoplasia by designing a model encompassing primary epithelial CCA organoids (CCAOs) and native liver and tumor scaffolds obtained by decellularization Background & Aims: Cholangiocarcinoma (CCA) is a hepatobiliary malignancy with dismal prognosis. Currently available models fail to recapitulate the full complexity of CCA, particularly the desmoplastic environment and the interplay between cancer cells and the extracellular matrix (ECM). We aimed to study the role of epithelial tumor cells in ECM deposition and desmoplasia by designing a model encompassing primary epithelial CCA organoids (CCAOs) and native liver and tumor scaffolds obtained by decellularization. Results: Decellularization resulted in effective removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin. When culturing CCAOs in CCA-M, compared to TFL-M and BME, the genome-wide gene expression profile much more resembled the transcriptome of primary CCA tumor tissue in vivo, with an accompanying increase in chemoresistance. CCAOs in decellularized matrix, both CCA-M and TFL-M, exhibited the formation of complex morphological structures, and revealed environment-dependent proliferation and migration dynamics, driven by the occurrence of epithelial-mesenchymal transition (EMT). CCA-M induced specific extracellular matrix protein production in CCAOs, such as fibronectin 1 (FN1), which is related to desmoplasia and patient survival. In TFL-M, lacking the desmoplastic environment, CCAOs were able to initiate a desmoplastic reaction directly through increased production of multiple collagen types. Conclusions: This improved model of cholangiocarcinoma, combining organoids and native extracellular matrix, recapitulates key components of CCA tumor biology, including transcriptome profiles, migration patterns, EMT, and ECM protein production. The increased production of extracellular matrix proteins suggests that epithelial tumor cells can contribute to their own desmoplastic environment.

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The confined environment in microfluidic reveals a hidden role of self-organizing extracellular-matrix in hepatic commitment and organoid formation of hiPSCs

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