Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.

Gonadotropin-releasing hormone (GnRH) neurons regulate puberty onset and sexual reproduction by secreting GnRH to activate and maintain the hypothalamic-pituitary-gonadal axis. During embryonic development, GnRH neurons migrate along olfactory and vomeronasal axons through the nose into the brain, where they project to the median eminence to release GnRH. The secreted glycoprotein SEMA3A binds its receptors neuropilin (NRP) 1 or NRP2 to position these axons for correct GnRH neuron migration, with an additional role for the NRP co-receptor PLXNA1. Accordingly, mutations in SEMA3A, NRP1, NRP2 and PLXNA1 have been linked to defective GnRH neuron development in mice and inherited GnRH deficiency in humans. Here, we show that only the combined loss of PLXNA1 and PLXNA3 phenocopied the full spectrum of nasal axon and GnRH neuron defects of SEMA3A knockout mice. Together with Plxna1, the human orthologue of Plxna3 should therefore be investigated as a candidate gene for inherited GnRH deficiency.

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc⁺HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc⁺HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.

β-cell replacement has been proposed as an effective treatment for some forms of diabetes, and in vitro methods for β-cell generation are being extensively explored. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of three regulators of pancreatic endocrine formation and β-cell identity, Ngn3, Pdx1 and MafA. Pancreatic ductal organoid cultures have recently been developed that can be expanded indefinitely, while maintaining the potential to differentiate into the endocrine lineage. Here, using mouse pancreatic ductal organoids, we see that co-expression of Ngn3, Pdx1 and MafA are required and sufficient to generate cells that express insulin and resemble β-cells transcriptome-wide. Efficiency of β-like cell generation can be significantly enhanced by preventing phosphorylation of Ngn3 protein and further augmented by conditions promoting differentiation. Taken together, our new findings underline the potential of ductal organoid cultures as a source material for generation of β-like cells and demonstrate that post-translational regulation of reprogramming factors can be exploited to enhance β-cell generation.