Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin β1-ILK-β-catenin-JUN axis.

[Display omitted] • Avidity with LM-E8 regulates the hematopoiesis potential of hPSCs maintained on. • LM-E8s affect the ITGB1-ILK-β-catenin-JUN axis for hematopoietic differentiation. • Higher ITGB1 levels indicate better hematopoietic potential of hPSCs. • Adding bFGF, SB431542 and heparin to the hPSC-sac method enhances HPC yields. • The improved method increases the definitive hematopoiesis of hPSCs. Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/β-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin β1 (ITGB1) and downstream integrin-linked protein kinase (ILK), β-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/β-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-β-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general. [ABSTRACT FROM AUTHOR]