Synergistic modulation of signaling pathways to expand and maintain the bipotency of human hepatoblasts

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

Robust expansion and functional maturation of human hepatoblasts by chemical strategy

“…Previous studies have demonstrated that stimulation of Wnt and inhibition of TGF-β were both important for hepatic proliferation [7,14,22]. Our recent study correlated with these reports and further demonstrated that Hedgehog signaling is also involved in stemness maintenance and proliferation of HBs [6]. Thus, we kept the TGF-β inhibitor A8301, Wnt agonist CHIR, and Hedgehog activator SAG, while replacing the remaining growth factors (EGF, HGF, and BMP4) with chemicals to optimize the HB culture condition.…”

“…In contrast, hPSCs-derived HBs show advantages over PHs and reprogramed HPLCs, owing to a wider and more convenient cell source, stronger proliferation ability, and more efficient differentiation into mature hepatocytes [6,8,20]. However, previous reported HB amplification formulations contained growth factors that were unsuitable for large-scale cell production.…”

“…Dihexa was identified in a screen that assessed the capacity to potentiate the biological activity of HGF and was originally developed as an antidementia drug [25]. Another signaling pathway that needed to be considered is the BMP signaling, which is the key driver of HBs, which was proved to be essential for the bipotency maintenance of HBs [6]. The absence of BMP signaling causes hepatic differentiation shifts to other lineages [26].…”

“…Human HB culture and hepatic maturation HBs were generated from hPSCs and maintained in expansion medium as described previously [6]. Briefly, the hPSCs were differentiated into HB and the Ep-CAM + /C-Kit − cells were purified by FACS from differentiation cultures and maintained on the growth factor reduced Matrigel (Corning).…”

“…HBs are capable of self-renewal and have the potency to differentiate into hepatocytes and hence expand purified HBs derived from hPSCs which would be an ideal strategy to enable massive hepatic cell production. Our previous study has established a cocktail (ABCEHS) to synergistically regulate BMP, Wnt, Hedgehog, and other signaling pathways that created a fine balance for HB expansion and bipotency maintenance [6]. Recent studies also reported hepatic expansion and maturation methods mostly applied expensive growth factors, which leads to critical challenges such as the high cost and protocol reproducibility in large-scale production [7][8][9].…”