Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is currently limited to post-termination samples. This hampers the prenatal investigation of fetal development and congenital diseases. Therefore, novel patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue or cellular reprogramming, would be advantageous. The amniotic fluid (AF) is a source of cells originating from multiple developing organs. Using single cell analysis, we characterised the cellular identities present in the human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids (AFO) exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modelling, we derived lung organoids from the amniotic and tracheal fluid cells of Congenital Diaphragmatic Hernia (CDH) fetuses. CDH organoids show differences to non-CDH controls recapitulating some features of the disease. AFO are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalised to the fetus at clinically relevant developmental stages.