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. In the last 5 years the signalling environment promoting human naïve pluripotency has been extensively investigated. However, little is known about the role exerted by the extracellular matrix and three-dimensional organization in naïve maintenance. Here, by using an unbiased and integrated approach we found that naïve, but not primed, hiPSC colonies are characterized by an extracellular matrix-rich microenvironment, and by a specific extracellular matrix fingerprint at transcriptional, proteomic and secretome levels. Based on this, we developed a 3D culture system which supports robust long-term feeder-free expansion of naïve hiPSCs. In this same 3D ECM-rich environment, we can recapitulate 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 three-dimensions starting from a single cell. Bulk RNA-seq. Microfluidic 3-day culture of human naïve iPSCs on MEF feeder layer (3 replicates) and on Vitronectin coating (3 replicates).