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

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 signalling pathways during maintenance of human naïve pluripotency have been characterized. However, little is known about the role exerted by the extracellular matrix (ECM) and its three-dimensional (3D) 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 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. Overall design: Bulk RNA-seq. Comparison of the transcriptomic profile of naïve hiPSCs cultured in 3D Matrigel, primed hiPSCs in 3D Matrigel, primed hiPSCs in 2D, and naïve hiPSCs cultured in 3D Matrigel undergoing priming. Naïve 3D hiPSCs and Primed 2D hiPSCs were harvested at day 4 of culture, primed 3D hiPSCs were harvested at day 5 of culture, while cells in priming condition were harvested at day 2, 4, 6, 8 and 10 of culture. Quadruplicates were collected for every condition and total RNA extracted with Qiagen RNeasy Mini Kit.