Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling

Abstract

Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.definitive endoderm ͉ function ͉ hepatocyte ͉ drug metabolism ͉ high throughput H uman embryonic stem cells (hESCs) are derived from the inner cell mass of preimplantation embryos and demonstrate pluripotency in vitro and in vivo (1, 2). Such attributes allow hESCs to be differentiated down all germ lineages in large numbers and offer significant advantages over their adult stem cell counterparts, which are generally limited in their capacity to differentiate and proliferate (3). Although these hESCs provide a valuable source of adult differentiated cells, homogeneous cellular differentiation to specific germ layers has proven difficult to achieve. One potential explanation for this failure is that cells do not receive sequential developmental cues that they do in vivo.Wnt signaling has been shown to play an important role in hESC self-renewal and differentiation and stimulates numerous intracellular signal transduction cascades, including the canonical pathway regulating gene expression in the nucleus and what seems to be a network of noncanonical pathways regulating many other aspects of cell biology [reviewed by Cadigan and Liu (4)]. In the absence of Wnt signaling, ␤-catenin is targeted for degradation; however, active Wnt signaling inhibits ␤-catenin destruction, resulting in its nuclear translocation (5, 6). After nuclear localization, ␤-catenin dimerizes with the nuclear proteins from the T cell factor/lymphoid enhancer factor (TCF/ LEF) family and transactivates gene expression. TCF/LEFs are not only present in transcriptional activator complexes; they also play a role in corepressor complex assembly (6).The important role played by Wnt signaling during gastrulation in vivo is evidenced by gene knockouts or dominant negatives (7, 8). Wnt3-mediated Brachyury expression is also important for migration of precursor cells through the anterior region of the primitive streak (PS). The subsequent specification of the anterior region of the PS to mesoderm or endoderm is likely to depend on the duration and magnitude of Nodal signaling (...

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

“…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.…”

Activin A Promotes Hematopoietic Fated Mesoderm Development Through Upregulation of Brachyury in Human Embryonic Stem Cells

“…In hESCs, however, this factor has been used to direct differentiation toward endoderm [22][23][24][25][26] and its potential action on the specification of hematopoietic mesoderm has yet to be reported. The inability of Activin A to stimulate the development of CD45 neg PFV hemogenic precursors combined with the reduction in frequency of hematopoietic output Bars for all panels indicate mean -SEM (*P < 0.05).…”

“…However, in instances where the activity of AF have been examined in hESCs, these factors have been shown to either maintain pluripotency [8,20,21] or induce endoderm specification [22][23][24][25][26] with one recent article examining their roles in early mesodermal differentiation in conjunction with bone morphogenetic protein 4 (BMP4) [27], but not hematopoiesis. Consequently, despite the documented role of AF in the regulation of human adult hematopoiesis [28,29], little focus has been directed to addressing the role of these factors in the progression of hESC development from mesoderm to blood.…”

Cellules souches embryonnaires humaines et iPS

“…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].…”

“…Elle est induite par l'ajout de HGF (hepatocyte growth factor) puis de dexaméthasone et d'oncostatine. Les cellules obtenues expriment de nombreux marqueurs hépatiques, par exemple CK8/18, AFP (−foetoprotéine), AAT (1-antitrypsine), HNF4 (hepatic nuclear factor) et montrent une certaine fonctionnalité in vitro comme le stockage du glycogène ou une faible activité de certains cytochromes P450 [2]. Toutefois, dans les rares cas où la fonctionnalité des cellules a été analysée in vivo, elle l'a été à très court terme, 5 jours après l'injection des cellules ; à plus long terme, les cellules ont entraîné la formation de tératomes voire d'adénocar-cinomes [2,4,6], probablement en raison de la persistance de CSEh indifférenciées parmi les cellules greffées.…”