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Objective
To investigate the mechanism of Wnt5a in lentoid body (LB) induction from human embryonic stem cells (hESCs).
Methods
A “three-stage” protocol was used for LB differentiation from hESCs in vitro, and Wnt5a level was modified by adding exogenous 500 ng/ml Wnt5a on day 18 as Wnt5a treatment group.Cells of control group and Wnt5a treatment group were collected on day 35.Cells were photographed by using the Zeiss Axio Observer Z1 microscope.Transcriptome sequencing was applied by Illumina.Genes with P value≤0.05 and fold change≥1.5 were identified as differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to determine the biological functions of DEGs.
Results
Compared with the control group, larger lentoid bodies were obtained in the Wnt5a treatment group.Transcriptome sequencing result showed that 478 genes were down-regulated and 201 genes were up-regulated in the Wnt5a treatment group compared with the control group, and Wnt5a up-regulated both lens cell differentiation and lens specific gene expression.Bioinformatics analysis result showed that most DEGs were involved in extracellular matrix remodeling, suggesting that Wnt5a regulated extracellular matrix remodeling during lens cell differentiation.The enrichment analysis result also showed that epithelial-to-mesenchymal transformation related processes were inhibited after Wnt5a treatment, suggesting that Wnt5a inhibited the abnormal differentiation of lens cells (especially lens epithelial cells) during lens cell differentiation.Wnt5a influenced the processes related to cytoskeleton remodeling.
Conclusions
Wnt5a may act in lens cells through MAPK/ERK signaling pathways to affect ECM and cell cytoskeletal organization, which provides a new direction for studying lens development.