Wnt5a Contributes to the Differentiation of Human Embryonic Stem Cells into Lentoid Bodies Through the Noncanonical Wnt/JNK Signaling Pathway

Abstract

Purpose: Recent work has indicated that Wnt5a has a critical role in embryonic development. We investigate whether the Wnt5a-activated noncanonical Wnt pathway is capable of promoting embryonic lens differentiation. Methods: A "three-stage" protocol was used to induce lens differentiation of human embryonic stem cells (hESCs) in vitro, and Wnt5a levels were modified by addition of exogenous protein and RNA interference. SP600125 was adopted to inhibit JNK cascades. The number and size of lentoid bodies obtained were measured, and quantitative RT-PCR, Western blotting, and immunofluorescence were used to detect gene and protein expression. Results: The quantity and size of lentoid bodies generated were significantly increased by addition of exogenous Wnt5a. Moreover, expression of lens-specific genes, including CRYAA, CRYAB, BFSP1, and MIP, and the lens fiber differentiation regulator PROX1 were prominently increased. We also observed activation of noncanonical Wnt signaling via upregulation of Dvl2, Rac1, and JNK. When Wnt5a-knockdown hESCs were induced to differentiate, fewer and smaller lentoid bodies resulted. In addition, expression of genes specific to lens was decreased and noncanonical Wnt/JNK pathway activity was downregulated. Accordingly, inhibition of JNK cascade suppressed the formation of lentoid bodies as well, consistent with that of Wnt5a-knockdown group. Conclusions: Wnt5a can promote the differentiation of hESCs into lentoid bodies through the noncanonical Wnt/JNK signaling pathway, thereby contributing to the study of human lens development and moreover the underlying etiology congenital cataracts.