The Microfluidic Environment Reveals a Hidden Role of Self-Organizing Extracellular Matrix in Hepatic Commitment and Organoid Formation of hiPSCs

Federica Michielin,Giovanni G Giobbe,Camilla Luni,Qianjiang Hu,Ida Maroni,Michael R Orford,Anna Manfredi,Lucio Di Filippo,Anna L David,Davide Cacchiarelli,Paolo De Coppi,Simon Eaton,Nicola Elvassore

doi:10.1016/j.celrep.2020.108453

Abstract

SummaryThe 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.

Highlights

The specification of cell identity during mammalian liver development relies on the activity of transcriptional networks
We found that when human pluripotent stem cells (hPSCs) are seeded at high density in mF in endoderm differentiation medium, the exit from pluripotency is restricted to few FOXA2+ cells that segregate from OCT4+ cells (Figure S1A)
When accumulation of endogenous factors is promoted, we obtained hepatocyte-like cells with higher Albumin secretion and cytochrome activity in a shorter period of time in mF compared to conventional culture conditions (CCC) (Giobbe et al, 2015)

Summary

Introduction

The specification of cell identity during mammalian liver development relies on the activity of transcriptional networks These networks are controlled by extrinsic signals that restrict and define distinct cell fates (Mamidi et al, 2018). From mouse in vivo studies, it is known that following the formation of the foregut endoderm, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) signaling from the surrounding mesoderm induce hepatic fate. In addition to FGF and BMP, hepatocyte growth factor (HGF) signaling from the septum transversum mesenchyme is required at this stage for hepatoblast proliferation and liver bud growth (Zorn, 2008; Si-Tayeb et al, 2010a), whereas the 3D assembly allows for the formation of proper polarity during maturation

Results

Discussion

Conclusion