Activin A Efficiently Specifies Definitive Endoderm from Human Embryonic Stem Cells Only When Phosphatidylinositol 3-Kinase Signaling Is Suppressed

Abstract

Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3-kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor ␤ family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast-conditioned medium under feeder-free conditions, ϳ70%-80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1-II. Microarray and quantitative polymerase chain reaction-based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin-like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self-renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC-DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self-renewal and early cell fate decisions. STEM CELLS 2007;25:29 -38

Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins

“…Differentiation of hPS cells to definitive endoderm under completely defined conditions remains challenging. Typical procedures involve exposure of hPS cells to activin A, combinations of growth factors, and/or undefined components like FBS or Matrigel (7,9,10). To determine the effects of the insoluble substratum on definitive endoderm differentiation, we used serum-free medium supplemented with activin A. HES cells (H9) were cultured on Matrigel or GBP surfaces and exposed to differentiation medium for 3 d. Interestingly, the expression of pluripotency markers POU5F1 (encodes Oct4) and SOX2 was down-regulated earlier and more drastically in cells cultured on GBP surfaces vs. Matrigel.…”

“…Matrigel is the most widely used substratum for hPS cell propagation (5) and differentiation (6)(7)(8)(9)(10)(11)(12); it is derived from mouse sarcoma cells, and although its principal components are laminin, collagen, and entactin, Matrigel consists of up to 1,800 different proteins-including encapsulated growth factors-whose levels vary significantly from batch to batch (13). Accordingly, the contributions of Matrigel-delivered signals to specific phenotypic outcomes-self-renewal or differentiation-are difficult to characterize or control.…”

Hepatic accumulation of Hedgehog-reactive progenitors increases with severity of fatty liver damage in mice

“…53 Mesenchymal-epithelial transitions are a characteristic of mesendodermal progenitors and their immediate progeny. 7,8 The former are capable of differentiating along various lineages and can generate various types of epithelial cells, 7,8 including hepatocytes. 10 The Hh pathway regulates the growth and differentiation of mesendodermal cells.…”

“…5,6 Less certain is whether adult livers also retain more primitive multipotent progenitors, similar to mesendodermal cells that give rise to hematopoietic, endothelial, adipocytic, smooth muscle-like, and epithelial cell types during embryogenesis. 7,8 Development of many tissues, including the liver, requires migration of the mesendoderm during gastrulation. 9 Recently, mesendodermal-like cells expressing the epithelial marker, EpCAM, were isolated from human fetal livers.…”

Cellules souches embryonnaires humaines et iPS

“…Cette étape est contrôlée par une combinaison de 4 facteurs, nécessaires mais suffisants, utilisée pendant 3 jours : concentration élevée d'activine A, de BMP4 et de FGF2, facteurs connus pour contrôler la spécification des 3 lignées germinales chez les mammifères, auxquels est ajouté du LY294004 (un inhibiteur chimique de la PI3K) [8]. Le milieu utilisé est un milieu défini (AFBLy) [9] • Spécification hépatique.…”