Comparable Generation of Activin-Induced Definitive Endoderm via Additive Wnt or BMP Signaling in Absence of Serum

Abstract

SummaryThere is considerable interest in differentiating human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and pancreatic cells for in vitro disease modeling and cell replacement therapy. Numerous protocols use fetal bovine serum, which contains poorly defined factors to induce DE formation. Here, we compared Wnt and BMP in their ability to cooperate with Activin signaling to promote DE formation in a chemically defined medium. Varying concentrations of WNT3A, glycogen synthase kinase (GSK)-3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime (BIO), and BMP4 could independently co-operate with Activin to effectively induce DE formation even in the absence of serum. Overall, CHIR99021 is favored due to its cost effectiveness. Surprisingly, WNT3A was ineffective in suppressing E-CADHERIN/CDH1 and pluripotency factor gene expression unlike GSK-3 inhibitors or BMP4. Our findings indicate that both Wnt and BMP effectively synergize with Activin signaling to generate DE from hPSCs, although WNT3A requires additional factors to suppress the pluripotency program inherent in hPSCs.

Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation

“…Furthermore, combining BMP4 and activin A can promote DE formation [8]. BMP4 and CHIR99021 have similar effects in promoting DE formation [20]. However, no synergetic effect was found when applying both factors together with activin A.…”

“…The ability of S17d5 to identify SOX17 + DE cells was assessed by differentiating cells with the first reported protocol established by D'Amour et al [6]. Due to the low bioactivity and high expense of using Wnt3a recombinant protein, it was replaced by a glycogen synthase kinase-3 (GSK-3) inhibitor, CHIR99021, in the D'Amour protocol [20]. This modified D'Amour protocol was termed Mod-D'Amour and used as a control while testing different modified protocols.…”

“…The GSK-3 inhibitor, CHIR99021, has been reported to support both DE and mesoderm differentiation from human PSCs through activation of Wnt signaling [20,27]. The specific cell fate is modulated by concentration and incubation time [27,28].…”

“…BMP4 represses cell pluripotency through BMP signaling [8,20]. With altered signaling time windows and interaction with different pathways, BMP4 has been reported to differentiate hESCs into various cell types.…”

Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids

“…It is activated by Nodal ligand binding with epidermal growth factor receptor-Cripto-FRL1-Cryptic co-receptor (Cripto) and Activin type I and II serine/threonine kinase receptors (ALK4 and ActRIIB, respectively), which results in phosphorylation of Smad2 and subsequent downstream gene regulation. [33] Activin A is a related TFG-β superfamily member, and can bind to nearly the same set of receptors as Nodal with the exception of Cripto. Although both Nodal and Activin A generate DE with similar global gene expression in vitro , Chen et al recently showed that Nodal-derived DE contributed to functionally superior adult tissues (liver and pancreas) in vivo compared to Activin-derived endoderm.…”