A Matrigel-Based Three-Dimensional Culture System for Organoid-Like Differentiation of Human iPS Cell-Derived Embryoid Bodies

Pluripotent stem cell differentiation is commonly evaluated using in vivo assays; however, these approaches are time-consuming, technically demanding, and limited in experimental controllability. To address these limitations, we aimed to establish a controllable in vitro platform for studying the multi-lineage differentiation of human induced pluripotent stem cells (iPSCs) in a three-dimensional (3D) environment. We developed a Matrigel-based 3D culture system for the organoid-like differentiation of human iPSC-derived embryoid bodies (EBs), hereafter referred to as a Matrigel-based in vitro 3D differentiation platform (MIVDP). To generate EBs, naïve-like iPSCs carrying enhanced green fluorescent protein (EGFP), introduced via a piggyBac-based transposon system prior to naïve-like conversion, were used. These EBs were then embedded in Matrigel and cultured on membrane filters at the air-liquid interface in a standard medium, such as Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, for up to three weeks. Under these conditions, EBs formed 3D multicellular structures containing various differentiated cell types derived from all three germ layers, as confirmed by histological and immunocytochemical analyses. Furthermore, when cultured in an osteogenic differentiation medium, EB-derived structures preferentially differentiated into osteoblast-like cells, as confirmed by Alizarin Red S staining and osteogenic marker gene expression. Because this system recapitulates certain aspects of multi-lineage differentiation and tissue organization, it can provide a controllable in vitro platform for studying the 3D differentiation and lineage specification of human iPSCs.