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 limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in 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 exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Primary human organoids models: Current progress and key milestones.

During the past 10 years the world has experienced enormous progress in the organoids field. Human organoids have shown huge potential to study organ development, homeostasis and to model diseases in vitro. The organoid technology has been widely and increasingly applied to generate patient-specific in vitro 3D cultures, starting from both primary and reprogrammed stem/progenitor cells. This has consequently fostered the development of innovative disease models and new regenerative therapies. Human primary, or adult stem/progenitor cell-derived, organoids can be derived from both healthy and pathological primary tissue samples spanning from fetal to adult age. The resulting 3D culture can be maintained for several months and even years, while retaining and resembling its original tissue's properties. As the potential of this technology expands, new approaches are emerging to further improve organoid applications in biology and medicine. This review discusses the main organs and tissues which, as of today, have been modelled in vitro using primary organoid culture systems. Moreover, we also discuss the advantages, limitations, and future perspectives of primary human organoids in the fields of developmental biology, disease modelling, drug testing and regenerative medicine.

Paediatric gastric organoids as a tool for disease modelling and clinical translation.

PURPOSE: Knowledge of gastric epithelial homeostasis remains incomplete, lacking human-specific models for study. This study establishes a protocol for deriving gastric epithelial organoids from paediatric gastric biopsies, providing a platform for modelling disease and developing translational therapies. METHODS: Full-thickness surgical samples and endoscopic mucosal biopsies were obtained from six patients. Gastric glands were isolated by a chemical chelation protocol and then plated in 3D culture in Matrigel® droplets in chemically defined medium. After formation, organoids were passaged by single cell dissociation or manual disaggregation. Cell composition and epithelial polarity of organoids were assessed by bright field microscopy and immunofluorescence analysis, comparing them to native paediatric gastric tissue. RESULTS: Gastric glands were successfully isolated from all six patients who were aged 4 months to 16 years. Gastric glands from all patients sealed to form spherical gastric organoids. These organoids could be passaged by manual disaggregation or single cell dissociation, remaining proliferative up to 1 year in culture. Organoids retained normal epithelial cell polarity, with the apical surface orientated towards the central lumen. Organoids expressed markers of mature gastric epithelial cell types, except for parietal cells. CONCLUSION: Gastric organoids can be reliably generated from paediatric biopsies and are a representative in vitro model for studying gastric epithelium.