Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells

Abstract

The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.

Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway

“…Because activin A-induced differentiation does not generate homogenous cell population (Supplementary information, Figure S1B), we used CD184 (also named CXCR4), a well-known definitive endoderm marker [4,[19][20][21], to sort the cell population and confirmed successful endoderm differentiation by RT-qPCR analysis (Supplementary information, Figure S1C). In addition to CD184, another report [20] used both CD184 and CD117 (also named c-KIT) to mark the definitive endoderm cell population. Interestingly, we found that CD184+ cells are almost always CD117+ in our differentiation system (Supplementary information, Figure S1D).…”

Nodal·Gdf1 Heterodimers with Bound Prodomains Enable Serum-independent Nodal Signaling and Endoderm Differentiation

“…Indirect Immunofluorescence Staining-Monolayer culture immunostaining was performed as described previously (2). The following primary and secondary antibodies were used: mouse anti-OCT3/4 (BD Transduction Laboratories; 1:500), rabbit anti-SOX17 (Beta Cell Biology Consortium; 1:500), donkey anti-mouse Alexa Fluor 647 (Invitrogen; 1:800), and donkey anti-rabbit Alexa Fluor 488 (Jackson ImmunoResearch Laboratories; 1:800).…”

“…6B). Because Nodal⅐Gdf1 had a higher specific activity than Nodal and was independent of serum, we compared its potential with that of commercially available Nodal and Activin A to differentiate pluripotent human ES cells into DE (2). First, treatment of hESCs with only Wnt3a and basic FGF already resulted in the down-regulation of the pluripotency marker OCT4 and significant up-regulation of DE markers such as SOX17 (Fig.…”

“…1). Despite the central role of Nodal in specifying endoderm during development, current protocols for directed in vitro differentiation of endoderm derivatives rely on the related ligand Activin A as a surrogate, presumably because Activin is more active than commercially available recombinant Nodal (2)(3)(4)(5)(6)(7)(8). However, a recent study showed that Nodal induces endoderm progenitors that are more competent to mature into functional insulin-producing cells if it is added to cultured mouse ES cells at a 20-fold higher concentration than Activin A (9).…”

“…Nodal assembles heteromeric complexes of the type I and II Activin receptors (Acvr1b 2 and Acvr2a or Acvr2b) that stimulate the phosphorylation and nuclear translocation of Smad2 and Smad3 transcription factors (10). Nodal is secreted as a precursor that must be cleaved by the proprotein convertases Furin or Pace4 to remove an N-terminal prodomain.…”