Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Abstract

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

“…It is well known that EBs consist of a cluster of three embryonic germ layers and an outer layer of primitive endoderm that produces α-fetoprotein (AFP) and GATA4 [26,27]. After 10 days of EB formation, many, but not all, of the control EBs formed three-dimensional aggregates, each with a distinguishable outer layer (Fig.…”

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

“…Instead, ERK signaling downstream from Fgf2 must accompany a feeder layer in serum-containing media for optimal hESC self-renewal . Interestingly, recombinant Fgf2 by itself could not replace feeders, and Fgf2 has been suggested to work in part by stimulating feeders to produce Activin/Nodal ligands; the combination of Fgf2 and Nodal/ Activin is sufficient to support hESC self-renewal in serum-free chemically defined culture conditions (Vallier et al 2004(Vallier et al , 2009James et al 2005).…”

Expression of the embryonic morphogen Nodal in cutaneous melanocytic lesions

“…4 In vitro, Nodal/ activin signaling maintains the pluripotency of human embryonic stem cells. [5][6][7] Although the functions of Nodal in embryogenesis are well known, its potential roles in postnatal physiology and disease are poorly understood. There is extensive evidence that TGF-b family signaling has roles both in tumor suppression and tumor progression in malignancies, including melanoma.…”