Abstract
BackgroundThe limited proliferative ability of hepatocytes is a major limitation to meet their demand for cell-based therapy, bio-artificial liver device, and drug tests. One strategy is to amplify cells at the hepatoblast (HB) stage. However, expansion of HBs with their bipotency preserved is challenging. Most HB expansion methods hardly maintain the bipotency and also lack functional confirmation.MethodsOn the basis of analyzing and manipulating related signaling pathways during HB (derived from human induced pluripotent stem cells, iPSCs) differentiation and proliferation, we established a specific chemically defined cocktails to synergistically regulate the related signaling pathways that optimize the balance of HB proliferation ability and stemness maintenance, to expand the HBs and investigate their capacity for injured liver repopulation in immune-deficient mice.ResultsWe found that the proliferative ability progressively declines during HB differentiation process. Small molecule activation of Wnt or inhibition of TGF-β pathways promoted HB proliferation but diminished their bipotency, whereas activation of hedgehog (HH) signaling stimulated proliferation and sustained HB phenotypes. A cocktail synergistically regulating the BMP/WNT/TGF-β/HH pathways created a fine balance for expansion and maintenance of the bipotency of HBs. After purification, colony formation, and expansion for 20 passages, HBs retained their RNA profile integrity, normal karyotype, and ability to differentiate into mature hepatocytes and cholangiocytes. Moreover, upon transplantation into liver injured mice, the expanded HBs could engraft and differentiate into mature human hepatocytes and repopulate liver tissue with restoring hepatocyte mass.ConclusionOur data contribute to the understanding of some signaling pathways for human HB proliferation in vitro. Simultaneous BMP/HGF induction, activation of Wnt and HH, and inhibition of TGF-β pathways created a reliable method for long-term stable large-scale expansion of HBs to obtain mature hepatocytes that may have substantial clinical applications.Graphical abstract
Highlights
The limited proliferative ability of hepatocytes is a major limitation to meet their demand for cellbased therapy, bio-artificial liver device, and drug tests
When human Induce pluripotent stem cells (iPSCs) reached nearly 70% confluence, mTeSR1 medium was replaced with differentiation medium (RPMI1640 [Gibco], supply with 1 × B27[minus insulin, Invitrogen]), containing 100 ng/mL Activin A (R&D Systems) and 3 μM CHIR99021 (CHIR) for 1 day, and on the following 2 days, CHIR was omitted from the medium
Flow cytometry analyses showed that more Ki67-positive cells (74.1% at day 3 and 60.3% at day 6) were observed at early stages. These Ki67-positive cells decreased in the HB stage (12% at day 10), which was consistent with the subdued cell growth during the differentiation process (Fig. 1c)
Summary
The limited proliferative ability of hepatocytes is a major limitation to meet their demand for cellbased therapy, bio-artificial liver device, and drug tests. Hepatocyte transplantation and bio-artificial liver support have been clinically evaluated as effective methods for compensation of lost liver function and an alternative to liver transplantation [1,2,3,4]. These two methods are hindered by the shortage of viable organ donation and dysfunction of hepatocyte in vitro culture [5]. An alternative strategy to meet the demand for functional hepatocyte supply would represent a major clinical advance. IPSC-derived hepatic cells are an ideal source for autologous cell-based therapy and extracorporeal artificial liver application
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