Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development

Abstract

Generation of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as an alternative to orthotopic liver transplantation. However, the generation of differentiated hepatocytes from hESCs remains a major challenge, especially using a method compatible with clinical applications. We report a novel approach to differentiate hESCs into functional hepatic cells using fully defined culture conditions, which recapitulate essential stages of liver development. hESCs were first differentiated into a homogenous population of endoderm cells using a combination of activin, fibroblast growth factor 2, and bone morphogenetic protein 4 together with phosphoinositide 3-kinase inhibition. The endoderm cells were then induced to differentiate further into hepatic progenitors using fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor. After further maturation, these cells expressed markers of mature hepatocytes, including asialoglycoprotein receptor, tyrosine aminotransferase, ␣1-antitrypsin, Cyp7A1, and hepatic transcription factors such as hepatocyte nuclear factors 4␣ and 6. Furthermore, the cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, cytochrome activity, and low-density lipoprotein uptake. After transduction with a green fluorescent protein-expressing lentivector and transplantation into immunodeficient uPA transgenic mice, differentiated cells engrafted into the liver, grew, and expressed human albumin and ␣1-antitrypsin as well as green fluorescent protein for at least 8 weeks. In addition, we showed that hepatic cells could be generated from human-induced pluripotent cells derived from reprogrammed fibroblasts, demonstrating the efficacy of this approach with pluripotent stem cells of diverse origins. Conclusion: We have developed a robust and efficient method to differentiate pluripotent stem cells into hepatic cells, which exhibit characteristics of human hepatocytes. Our approach should facilitate the development of clinical grade hepatocytes for transplantation and for research on drug discovery. (HEPATOLOGY 2010;51:1754-1765

Sustained Delivery Growth Factors with Polyethyleneimine‐Modified Nanoparticles Promote Embryonic Stem Cells Differentiation and Liver Regeneration

“…To date, numerous studies in rodents have shown that predifferentiated ESC transplantation can reverse acute fulminant hepatic failure 6,50, 51, 52, 53, 54. In our study, we employed day‐3‐induced DE cells as a source of isolated cells for transplantation into injured liver.…”

“…First, ESCs could generate functional hepatocytes in vivo that are then engrafted efficiently within the host liver 6. A study reported that 2.5%–5% is necessary for reversing the liver injuries 59.…”

“…Both murine and human ESCs were reported to differentiate into hepatocytes or hepatocyte‐like cells successfully 3, 4, 5, 6, 7. As known, most of the current protocols used embryoid bodies (EBs) and simple adherent monolayer cultures with soluble growth factors and small chemicals to differentiate ESCs into hepatocytes; however, they are still subjected to inefficient differentiation and low purity of functional hepatocytes 8, 9.…”

Cellules souches embryonnaires humaines et iPS

“…Celle-ci utilise des conditions chimiquement définies et suit les étapes clefs du développement hépatique [7] (Figures 1 et 2). Les trois étapes sont identiques à celles précédemment définies.…”

“…Nous avons montré que les conditions de différenciation des CSEh en hépatocytes que nous avons décrites permettent d'induire la différen-ciation d'iPS dérivées de fibroblastes humains en cellules de l'endoderme [29] puis en progéniteurs hépatiques [7]. Ainsi des études de fonctionnalité in vivo pourront être réalisées de façon comparative avec ces deux types de progéniteurs.…”