Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development In Vivo

Hay, David C.; Zhao, Debiao; Fletcher, Judy; Hewitt, Zoë; McLean, Doris; Urruticoechea‐Uriguen, Alai; Black, James R.; Elcombe, Cliff; Ross, James A.; Wolf, C. Roland; Cui, Wei

doi:10.1634/stemcells.2007-0718

Cited by 398 publications

(386 citation statements)

References 37 publications

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“…30) ES cells are efficiently differented to hepatocytes by priming ES cells towards definitive endoderm with activin A, that is a member of transforming growth factor-b (TGF-b) superfamily, prior to further differentiation to hepatocytes. 31) EBs formed by culture for 2 d can be successfully induced to differentiate into definitive endoderm by culture in activin A and FGF-2. 32) We do not know the reason why the expression levels of hepatocyte marker genes, especially ALB, in the cells cultured for 2 d for EB formation from cmES cells was higher than those in cells cultured for 5 d (Fig.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of Monkey Embryonic Stem Cells into Hepatocytes and mRNA Expression of Cytochrome P450 Enzymes Responsible for Drug Metabolism: Comparison of Embryoid Body Formation Conditions and Matrices

Momose

Matsunaga

Murai

et al. 2009

Biological & Pharmaceutical Bulletin

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Examination of drug metabolism using human hepatocytes is important in the early stages of drug development. However, primary human hepatocytes are short-lived and cannot be maintained in culture over the long term. Considerable donor-dependent variations are also problematic. Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts and are capable of differentiating into three embryonic germ layers and germ cells.1) The cells apparently differentiate into various types of mature cells, and are thereby an attractive source for routine access to large numbers of cells that can be used for the development of candidate drug-screening strategies replacing primary cells.2) However, ethical considerations have limited the availability of human ES cells. The phenotype of human ES cells is known to be similar to that of monkey ES cells but differs from that of mouse ES cells with regard to morphology, response to leukemia inhibitory factor, and gene expression patterns. 1,[3][4][5] Research using monkey ES cells is considered to be useful for investigation of differentiation mechanisms in primate ES cells and models of human ES cells. Recently, it has been shown that monkey ES cells can be differentiated into various cell types-including neurons, hematopoietic cells, and pancreatic cells-using growth factors. [6][7][8] Hepatocytes derived from monkey ES cells may be useful for pharmacokinetic examinations such as induction of drug metabolism enzymes and interactions of candidate drugs. To date however, there have been few reported studies describing the differentiation of monkey ES cells into hepatocytes. 9,10) After the emergence of the liver bud from the developing gut tube, the level of hepatic maturation is characterized by the expression of liver-and stage-specific genes. For example, alfa-fetoprotein (AFP) is an early hepatic marker, expressed by hepatoblasts in the liver bud until birth.11,12) The synthesis of AFP decreases dramatically after birth and only trace amounts are expressed in the adult liver. In contrast, albumin (ALB), the most abundant protein synthesized by hepatocytes, is initially expressed at lower levels in early fetal hepatocytes but this increases as the hepatocytes mature, reaching a maximum in adult hepatocytes. 13,14) The mRNA expression of cytochrome P450 7A1 (CYP7A1) which is a rate-limiting enzyme in the conversion of cholesterol to bile acids in liver 15) is detected in fetal liver of third trimester of pregnancy.16) After birth, CYP7A1 increases several-fold with age both at the enzyme activity level and the mRNA level. 17,18) The in vitro approaches involve the formation of embryoid bodies (EBs) to mimic the inductive microenvironment required for liver organogenesis [19][20][21] and treatment with specific growth factors and cytokines critical for hepatic differentiation.22) At present, culture systems for ES cells have mainly used gelatin-or collagen-coated plates as the matrix for the maintenance of cells in an undifferentiated state an...

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Section: Discussionmentioning

confidence: 99%

Differentiation of Monkey Embryonic Stem Cells into Hepatocytes and mRNA Expression of Cytochrome P450 Enzymes Responsible for Drug Metabolism: Comparison of Embryoid Body Formation Conditions and Matrices

Momose

Matsunaga

Murai

et al. 2009

Biological & Pharmaceutical Bulletin

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“…The contributing activin/ TGF-β ligands, however, have not been fully identified. Several studies have used activin A as part of protocols to differentiate human embryonic stem cells (hESC) into hepatocyte-like cells [62][63][64][65] . Like activin beta A, the beta B subunit is expressed in multiple tissues and organs [11,66] .…”

Section: Dysfunctionmentioning

confidence: 99%

Activins and follistatins: Emerging roles in liver physiology and cancer

Kreidl

Öztürk²,

Metzner³

et al. 2009

WJH

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Activins are secreted proteins belonging to the TGF-β family of signaling molecules. Activin signals are crucial for differentiation and regulation of cell proliferation and apoptosis in multiple tissues. Signal transduction by activins relies mainly on the Smad pathway, although the importance of crosstalk with additional pathways is increasingly being recognized. Activin signals are kept in balance by antagonists at multiple levels of the signaling cascade. Among these, follistatin and FLRG, two members of the emerging family of follistatin-like proteins, can bind secreted activins with high affinity, thereby blocking their access to cell surface-anchored activin receptors. In the liver, activin A is a major negative regulator of hepatocyte proliferation and can induce apoptosis. The functions of other activins expressed by hepatocytes have yet to be more clearly defined. Deregulated expression of activins and follistatin has been implicated in hepatic diseases including inflammation, fibrosis, liver failure and primary cancer. In particular, increased follistatin levels have been found in the circulation and in the tumor tissue of patients suffering from hepatocellular carcinoma as well as in animal models of liver cancer. It has been argued that up-regulation of follistatin protects neoplastic hepatocytes from activin-mediated growth inhibition and apoptosis. The use of follistatin as biomarker for liver tumor development is impeded, however, due to the presence of elevated follistatin levels already during preceding stages of liver disease. The current article summarizes our evolving understanding of the multi-faceted activities of activins and follistatins in liver physiology and cancer.

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“…Elle est contrôlée par l'ajout de molécu-les destinées à rendre les progéniteurs endodermiques sensibles aux signaux inducteurs de la différenciation hépatique. Ces molécules peuvent être des cytokines, telles Wnt3a [2], FGF4/BMP2 (fibroblast growth factor/bone morphogenic protein) [3] ou BMP4/FGF2, et elles sont généralement couplées soit à la culture en conditions hypoxiques [4] soit au sodium butyrate, inhibiteur de l'acétylation des histones [5], ou au DMSO (diméthylsulfoxide) induisant la méthylation de l'ADN [6].…”

Section: Différenciation Hépatique Des Cellules Esunclassified

Cellules souches embryonnaires humaines et iPS

2010

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Le foie est le siège de nombreux déficits métaboli-ques (maladies du cycle de l'urée, hypercholestéro-lémie familiale, syndrome de Crigler-Najjar type I, etc.), mais aussi la cible d'atteintes hépatiques chroniques ou aiguës. Actuellement la transplantation > La transplantation d'hépatocytes est considérée comme une alternative à la transplantation d'organes pour le traitement de maladies métaboliques notamment. Cependant, en raison des difficultés de disposer d'un grand nombre d'hépatocytes, de nouvelles sources de cellules ont été envisagées. Ces cellules peuvent être d'origine hépatique (cellules souches hépatiques) ou extra-hépatique, telles les cellules souches mésenchymateuses ou les cellules souches pluripotentes (cellules souches embryonnaires humaines [CSEh] ou iPS). Nous avons mis au point une méthode de diffé-renciation des CSEh en hépatocytes foetaux. Les conditions de différenciation qui reproduisent les étapes majeures du développement embryonnaire du foie sont établies en milieu défini, sans ajout de produit d'origine animale ou indéterminée. Les cellules obtenues expriment de nombreux marqueurs d'hépatocytes foetaux (cytochrome p450 3A7, albumine, alpha-1-antitrypsine, etc.). Elles sont capables d'assurer des fonctions spécifiques des hépatocytes (métaboliser l'ammonium, excré-ter le vert d'indocyanine), et enfin de se dévelop-per et d'exprimer des protéines hépatiques deux mois après transplantation dans le foie de souris nouveau-nés immunodéficientes (uPAxrag2gc -/-). Nous avons également démontré que ce protocole s'appliquait également aux iPS, et les études dans les modèles animaux nous permettront de comparer le potentiel in vivo de ces deux sources de cellules pluripotentes. Seules des approches précliniques effectuées chez le primate permettront de valider une éventuelle application à l'homme. < ortho topique est le seul traitement curatif des maladies métaboliques sévères engageant le pronostic vital mais elle est restreinte par la pénurie croissante de donneurs d'organe. La thérapie cellulaire apparaît depuis quelques années comme une alternative thérapeutique pour le traitement de ces maladies. En effet, elle permettrait de remplacer les seuls hépatocytes porteurs d'un défaut génétique, en laissant intact le foie. L'obstacle majeur de cette approche est que les hépatocytes ne peuvent pas être amplifiés en culture et résistent mal à la congélation. Il est donc nécessaire d'évaluer le potentiel d'autres sources de cellules, qui non seulement pourraient se différencier en hépatocytes mais aussi proliférer in vitro. Ces deux propriétés caractérisent les cellules souches pluripotentes, et en particulier les cellules souches embryonnaires et les iPS (induced pluripotent stem cells). Nous faisons le point dans cette revue sur les progrès réalisés dans l'obtention d'hépatocytes fonctionnels à partir de cellules souches pluripotentes en nous fondant sur l'expérience de notre équipe dans ce domaine.

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Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development In Vivo

Cited by 398 publications

References 37 publications

Differentiation of Monkey Embryonic Stem Cells into Hepatocytes and mRNA Expression of Cytochrome P450 Enzymes Responsible for Drug Metabolism: Comparison of Embryoid Body Formation Conditions and Matrices

Differentiation of Monkey Embryonic Stem Cells into Hepatocytes and mRNA Expression of Cytochrome P450 Enzymes Responsible for Drug Metabolism: Comparison of Embryoid Body Formation Conditions and Matrices

Activins and follistatins: Emerging roles in liver physiology and cancer

Cellules souches embryonnaires humaines et iPS

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