Abstract: To develop three-dimensional lentoid bodies (3D-LBs) with double-sided convexity and complete capsule wrapping that highly mimics the morphology and structure of the natural human lens.Polyacrylamide hydrogels of 1, 5, 20, and 100 kPa stiffness were prepared, and urinary human induced pluripotent stem cells (UiPSCs) were implanted on the hydrogels and induced into 3D-LBs by the “Fried egg” differentiation method. The key mechanistic signaling pathways affecting the differentiation were explored by transcriptome sequencing, adjusting the extracellular matrix content, YAP inhibitor, and ROCK inhibitor. The morphology, composition and molecular expression characteristics of the organoids were depicted with the help of hyperfield microscopy, confocal 3D fluorescence imaging, transmission electron microscopy, and qPCR. 3D-LBs, which were as convex as the bottom and top surfaces and were encapsulated by the collagen membrane, could be obtained on 1 kPa hydrogels. And it could advance the maturation time from 25 days for hard substrates to 12 days for soft substrates. Lens fiber differentiation of 1kPa-LB was rapidly activated after day 9, and transcriptome sequencing revealed significant downregulation of the extracellular collagen-related pathway and the Hippo pathway prior to fiber differentiation. 1% relatively low extracellular collagen concentration and transient inhibition of the ROCK/Hippo pathway were both beneficial for a more rapid differentiation of LBs.The biconvex 3D-LBs were successfully cultured on 1kPa hydrogels and greatly reduced the differentiation maturation time. The low stiffness mechanical environment may promote lens fiber differentiation by reducing extracellular collagen and inhibiting the ROCK/Hippo pathway early in development.
