Three-Dimensional Trilineage Differentiation Conditions for Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) hold great potential for regenerative medicine. However, optimizing their differentiation into specific lineages within three-dimensional (3D) scaffold-based culture systems that mimic in vivo environments remains challenging. This study examined the trilineage differentiation of hiPSCs under various 3D conditions using synthetic peptide hydrogel matrices with and without embryoid body (EB) medium induction. hiPSC 3D colonies (spheroids), naturally formed from single cells or small clusters in 3D culture, were used for differentiation into the three germ lineages. Differentiated spheroids exhibited distinct morphological characteristics and significantly increased expression of key lineage-specific markers-<i>FOXA2</i> (endoderm), <i>Brachyury</i> (mesoderm), and <i>PAX6</i> (ectoderm)-compared to undifferentiated controls. Marker expression varied depending on the 3D culture conditions. Differentiation efficiency improved significantly, increasing from 16% to 71% for endoderm, 61% to 80% for mesoderm, and 35% to 48% for ectoderm, by selecting the appropriate 3D matrix and applying EB induction. Comprehensive data analysis from RT-qPCR, immunocytochemistry staining, and flow cytometry confirmed that the Synthegel Spheroid (SGS) is a viable 3D matrix for evaluating all three germ lineages using a commercial trilineage differentiation kit. While EB induction is essential for endodermal differentiation, it is not required for mesodermal and ectodermal lineages. These findings are valuable not only for screening initial differentiation potential at the lineage level but also for optimizing 3D differentiation protocols for deriving somatic cells from hiPSCs.