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

Despite advances in prenatal diagnosis, it is still difficult to predict severity and outcomes of many congenital malformations. New patient-specific prenatal disease modelling may optimise personalised prediction. We and others have described the presence of mesenchymal stem cells in amniotic fluid (AFSC) that can generate induced pluripotent stem cells (iPSCs). The lengthy reprogramming processes, however, limits the ability to define individual phenotypes or plan prenatal treatment. Therefore, it would be advantageous if fetal stem cells could be obtained during pregnancy and expanded without reprogramming. Using single cell analysis, we characterised the cellular identities in amniotic fluid (AF) and identified viable epithelial stem/progenitor cells of fetal intestinal, renal and pulmonary origin. With relevance for prenatal disease modelling, these cells could be cultured to form clonal epithelial organoids manifesting small intestine, kidney and lung identity. To confirm this, we derived lung organoids from AF and tracheal fluid (TF) cells of Congenital Diaphragmatic Hernia (CDH) fetuses and found that they show differences to non-CDH controls and can recapitulate some pathological features of the disease. Amniotic Fluid Organoids (AFO) allow investigation of fetal epithelial tissues at clinically relevant developmental stages and may enable the development of therapeutic tools tailored to the fetus, as well as to predicting the effects of such therapies.