Abstract
The human primed pluripotent state is maintained by a complex balance of several signaling pathways governing pluripotency maintenance and commitment. Here, we explore a multiparameter approach using a full factorial design and a simple well-defined culture system to assess individual and synergistic contributions of Wnt, FGF and TGFβ signaling to pluripotency and lineage specification of human induced pluripotent stem cells (hiPSC). Hierarchical clustering and quadratic models highlighted a dominant effect of Wnt signaling over FGF and TGFβ signaling, drawing hiPSCs towards mesendoderm lineages. In addition, a synergistic effect between Wnt signaling and FGF was observed to have a negative contribution to pluripotency maintenance and a positive contribution to ectoderm and mesoderm commitment. Furthermore, FGF and TGFβ signaling only contributed significantly for negative ectoderm scores, suggesting that the effect of both factors for pluripotency maintenance resides in a balance of inhibitory signals instead of proactive stimulation of hiPSC pluripotency. Overall, our dry-signaling multiparameter modeling approach can contribute to elucidate individual and synergistic inputs, providing an additional degree of comprehension of the complex regulatory mechanisms of human pluripotency and commitment.
Highlights
Human induced pluripotent stem cells have an incredible potential for regenerative medicine therapies, drug-screening and disease modeling [1,2,3]
To expose the impact of FGF signaling, TGF/Nodal signaling and Wnt signaling in human pluripotency and exit towards differentiation, a full factorial design was conceived to detect dual signaling roles by combining three concentration levels of each signaling input: Zero, lower activation (1/3 of higher activation) and higher activation, using E6/VTN [15], a dry-signaling system, as the basal culture medium (Figure 1)
Both TeSR and E8 medium use 100 ng/mL of FGF2 to maintain human induced pluripotent stem cells (hiPSC) pluripotency [15,42]. At this concentration and higher, a plateau of maximal activity is observed for downstream FGF signaling targets such as ERK and FRS-2 [12]. Maximum activation of both downstream targets can be observed at 10 ng/mL, which can contribute to the pleiotropic behavior of FGF signaling [12]
Summary
Human induced pluripotent stem cells (hiPSCs) have an incredible potential for regenerative medicine therapies, drug-screening and disease modeling [1,2,3]. FGF, TGFβ and Wnt signaling pathways are among the most important pathways controlling hiPSC fate [4,5,6] These signaling pathways can be associated with pleiotropic effects, stimulating divergent cellular responses such as self-renewal and commitment [12,13,14]. Noteworthy is the fact that Wnt signaling has a role in directing cells from neuroectoderm towards neural crest specification [25,26], and that it inhibits cardiac mesoderm specification [27,28] while promoting the epicardial cell fate [29] These signaling pathways can be interconnected and influenced by multiple signals at different pathway nodes, resulting in synergistic or antagonistic effects that can shift commitment towards specific lineages [30,31,32,33]. FGF and TGFβ signaling contributed negatively to the ectoderm model without a significant contribution for the pluripotency model, suggesting that a balanced inhibitory effect is promoting hiPSC pluripotency maintenance
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
