Wnt Signaling Orchestration with a Small Molecule DYRK Inhibitor Provides Long-Term Xeno-Free Human Pluripotent Cell Expansion

Abstract

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-β (TGFβ) signaling pathways, and none provide for replacement of FGF/TGFβ ligands with chemical compounds. The Wnt/β-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however, the role of Wnt/β-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/β-catenin signaling in mouse embryonic stem cells, we identified a compound, ID-8, that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8, we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFβ activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.

p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)

“…This paradigm appears to be generalizable to both epithelial and non-epithelial adult somatic stem/progenitor cells. In support of the universality of this model, similar ␤-catenin/co-activator interactions have been shown to regulate differentiation of other stem/progenitor cells including mouse and human embryonic stem cells (9,34), cardiovascular progenitors (35,36), neuronal progenitors (37), hepatoblasts and hepatic tumor initiating cells (38), and leukemia cells (39). This report extends these studies to elucidate a novel intersection between non-canonical and canonical signaling arms, further highlighting the complexity of Wnt signaling.…”

Telomerase-mediated telomere elongation from human blastocysts to embryonic stem cells

“…The expression of Wnt family members in feeder cells was reported to enhance proliferation and improve survival in ESCs (Hasegawa et al, 2012). In addition, Wnt-b-catenin signaling has been linked to TERT expression in both mESCs and human carcinoma cells (Hoffmeyer et al, 2012).…”

Animal‐cell culture media: History, characteristics, and current issues

“…These cells initially were cultivated on a layer of feeder cells in a serum‐ or KSR‐supplemented medium,104 but in anticipation of clinical applications, the efforts shifted to the development of culture conditions that are feeder‐free and xeno‐free (Table 5). 105, 106, 107, 108, 109, 110, 111 Among these, the E8 medium that was developed by Guokai Chen et al. has become popular.…”