3D ECM-rich environment sustains the identity of naïve human iPSCs II

The establishment of in vitro naïve human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways during maintenance of human naïve pluripotency has been characterized. However, little is known about the role exerted by the extracellular matrix and its three-dimensional organization. Here, using an unbiased and integrated approach combining transcriptional, proteomic and secretome analyses, we found that naïve, but not primed, hiPSC colonies are characterized by a self-organized extracellular matrix (ECM)-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naïve hiPSCs, and also allows direct and timely developmental morphogenesis simply by modulating the signalling environment. Our study opens new perspectives for future applications of naïve hiPSCs to study critical stages of human development in 3D starting from a single cell Comparison of the transcriptomic profile of human naïve iPSCs cultured on feeders or in 3D matrigel (feeder-free). At passage number 10, cells were harvested and a cell sorting was performed to obtain both the bulk population and SUSD2+-only cells. 4 replicates per each sample (bulk or SUSD2+) were collected and total RNA extracted with Qiagen RNeasy Mini Kit.