Activin and BMP4 Synergistically Promote Formation of Definitive Endoderm in Human Embryonic Stem Cells

Abstract

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic b cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.

The crucial role of Activin A on the formation of primordial germ cell-like cells from skin-derived stem cells in vitro

“…The most common finding was that ActA can promote the formation of definitive endoderm in a defined differentiation medium, and it works with other proteins such as BMP4, a very important factor in germ cell fate determination during early embryonic development. 58,59 In human, ActA plus BMP4 strongly increased germ cell differentiation of hESC. 60 It was in accord with our finding that EBLC differentiation with BMP4 increased PGCLCs formation by ActA supplement.…”

The PDZ domain protein Mcc is a novel effector of non-canonical Wnt signaling during convergence and extension in zebrafish

“…Knockdown of MCC impairs lamellipodia formation and cell migration in Boyden chamber assays (Arnaud et al, 2009;Pangon et al, 2010Pangon et al, , 2012. Lastly, we recently observed enriched MCC expression in a microarray screen comparing the differentiation of human embryonic stem cells in response to combinations of TGFβ-related growth factors (Teo et al, 2012). High levels of MCC coincided with the expression of cardinal gastrulation marker genes and, in the mouse embryo, Mcc is specifically expressed in the primitive streak -the site of epithelial-to-mesenchymal transformation in the pluripotent epiblast, where nascent mesoderm and definitive endoderm emerge during gastrulation (Young et al, 2011).…”

The SMAD2/3 corepressor SNON maintains pluripotency through selective repression of mesendodermal genes in human ES cells

“…Differentiation was carried out according to the schematic in Figure 1A (Teo et al 2012) and, expectedly, resulted in the loss of pluripotency markers (NANOG, OCT4, and SOX2) and up-regulation of PS (WNT3, FGF8, EOMES, and MIXL1), mesendoderm (GSC and LHX1), and DE (FOXA2 and SOX17) markers by day 5 ( Supplemental Fig. 1A).…”

“…For feeder-free culture, HES3 and S4TR5 cells (Zafarana et al 2009) were maintained in mTeSR1 medium (Stem Cell Technologies) on 1:200 GFRMatrigel-coated (Becton Dickinson) dishes. DE differentiation was carried out as previously described (Teo et al 2012). …”