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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., The thymus is essential for T cell maturation and selection, and thymic defects result in severe immune problems. Here the authors identify a thymus cell population that is expandable in vitro, and can repopulate natural thymic matrix to generate tissue that supports mature T cell development in vitro and in vivo.

Liver fibrosis is driven by progressive accumulation of extracellular matrix (ECM), coupled with chronic inflammation. Recent evidence shows that ECM changes in liver disease directly and indirectly modulate the immune system. Since traditional cell culture models often lack immune cell and ECM components, bioengineered models of liver disease using decellularised tissues present an appealing alternative. Here, we describe the generation of a bioengineered model which incorporates the dynamic culture of circulating immune cells with decellularised human liver scaffolds, supported in a custom-made bioreactor to explore interactions between the immune system and liver ECM proteins at different stages of fibrosis. Liver ECM-scaffolds were generated bydecellularisingliver tissue with/without underlying disease from human liver tissue samples from partial hepatectomy, or by perfusion decellularisation of whole rat liver using established protocols. PBMCs isolated from healthy donor whole-blood were first cultured in the bioreactor under semi-continuous perfusion at high (4.22 Pa) or low shear stress (0.34 Pa) and compared to static conditions. Longitudinal profiling of PBMC phenotype was determined byFACs. ECM-scaffold were then perfused with PBMCs and cultured for 5 days. Immune-perfused tissues were stained for PBMC surface markers (T, B, NK cells and macrophages) to identify cell homing. We designed a custom-made bioreactor which can house 6 decellularised human liver scaffolds and which supports dynamic culture of PBMCs. We found that shear stress impacts PBMC viability and that PBMCs are better supported under low shear stress conditions. PBMC phenotype profiling byFACs show no significant changes in proportion of T cells between static and dynamic culture, while an increase in proportion ofTregsand a decrease in proportion of NK cells were detected in dynamic culture. Perfusion of PBMCs in decellularised scaffolds without fibrosis show homing of cells in different areas of the scaffold at all time points, with CD3+ T cells representing 50% of cells and with small clusters of macrophages, monocytes and B cells at 3 days of culture. Dynamic perfusion improved cell density and homing in decellularised liver scaffolds compared to static conditions. Our bioengineered system supports dynamic co-culture of PBMCs and decellularised scaffolds, with varying grades of fibrosis, allowing us to explore the interactions between the ECM and immune cells in liver disease in a dynamic perfusion system. A better understanding of ECM-immune interactions will aid our understanding of the driving factors behind fibrosis and could highlight new disease progression biomarkers and therapeutic targets to treat disease.

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., Organoid cultures have been developed from multiple tissues, opening new possibilities for regenerative medicine. Here the authors demonstrate the derivation of GMP-compliant hydrogels from decellularized porcine small intestine which support formation and growth of human gastric, liver, pancreatic and small intestinal organoids.

Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children1. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy.

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.

Background Irreversible intestinal failure (IIF) is a condition that can lead to significant morbidity and mortality and occurs as a result of anatomical or functional loss of the intestine. Many patients eventually require an intestinal transplant, which is unfortunately limited by poor survival (65% 1 year survival) and a scarcity of donor organs. Tissue engineering is an rapidly advancing field which we believe represents a highly promising potential solution to this problem. Stem cells isolated from the crypt of the intestine are an ideal cell source for this approach. However, they currently require 3D cell expansion in a commercially available hydrogel, Matrigel, which is currently not approved for clinical use. This represents a major road-block to the clinical translation of this otherwise extremely promising treatment option. To this end, our group have developed and optimised an extracellular matrix (ECM) hydrogel that is derived from decellularised intestinal tissue which has the potential to act as a Matrigel alternative. Method Piglet intestine is decellularised and formed into a gel using protocols developed by our group. These protocols were characterised using scanning electron microscopy (SEM), histology, immunohistochemistry, DNA and ECM quantification, rheology, spectrophotometry, angiogenic assays and proteomics. Cytocompatibility of intestinal stem cells in the gel was assessed with daily imaging, quantification and immunofluorescence. Results Results showed that a stable reproducible non-immunogenic gel is formed within 20 min post-application to 37°C, without the aid of any gelation agents. Proteomics showed a collagen-rich matrix with a plethora of other components including fibronectin, laminin myosin and fibrillin. Cell experiments demonstrated that the gel is biocompatible, non-cytotoxic and can facilitate cell viability with phenotypic morphology. Conclusion An ECM hydrogel derived from decellularised tissue represents a valuable tool for use in a many tissue engineering applications, primarily for 3D culturing of intestinal organoids. This discovery greatly widens the clinical potential for intestinal tissue engineering.

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. ispartof: NANOMATERIALS vol:11 issue:2 ispartof: location:Switzerland status: published