A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs

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

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

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

Investigating human pancreas development in a dish

Efforts are underway to develop cell therapies for diabetes using endocrine cells derived in vitro from human pluripotent stem cells. However, protocols are hampered by a scarcity of knowledge on human pancreatic development and on how to model endocrine differentiation of pancreatic progenitors in vitro. In this thesis, gene expression, Fluorescence Activated Cell Sorting (FACS) and immunohistochemical analyses were used, along with other techniques, to provide systematic characterisation of pancreatic cell populations during ontogenesis. Human fetal pancreatic progenitor cells (hPPCs) were successfully isolated from pancreatic tissue of different embryonic and fetal stages. hPPCs were extensively cultivated in 3D MatrigelTM cultures in Expansion Medium (EM), and in Organogenesis Medium (OM), to assess their multipotent nature. Freshly isolated or expanded hPPCs were triggered to differentiate into endocrine cells; notably a significant increase of endocrine markers was observed at gene and protein levels, even after extensive expansion. 3D organoid cultures rely mostly on MatrigelTM, a hydrogel derived from mouse sarcoma which is not suitable for clinical application. To overcome this, hPPC cultures were established utilizing fully synthetic, functionalised hydrogels with defined physical properties and also by producing hydrogels from native extracellular matrix (ECM). Cell growth and differentiation potency were modulated by ECM properties, opening the possibility of directing cell fate by integrating soluble factors, matrix components and cell-cell interaction mechanisms. In conclusion, this thesis provides a comprehensive characterisation of human embryonic and fetal pancreas, and addresses human pancreatic progenitor potency in clinically relevant culture conditions

stato protrombotico e attivazione piastrinica nell'ipertensione essenziale: implicazioni per il trattamento antiaggregante

L’ipertensione essenziale è definita come una condizione di elevata pressione sanguigna nella quale le cause secondarie, quali malattia renovascolare, insufficienza renale, feocromocitoma, aldosteronismo o altra cause di ipertensione secondaria o forme mendeliane (monogeniche) non sono presenti. Le cause di questa patologia quindi restano tutt’ora sconosciute e si ritiene sia un disordine eterogeneo nel quale sono coinvolti sia fattori genetici che ambientali. Essa rappresenta il 95% di tutti i casi di ipertensione e la prevalenza aumenta con l’età. Interessa il 25-30% della popolazione adulta e fino al 60-70% degli ultrasettantenni. Le principali complicanze dell’ipertensione, infarto miocardico ed ictus ischemico, sono di natura trombotica piuttosto che emorragica, fenomeno conosciuto come “paradosso pro-trombotico” o “Birmingham paradox”. Questa condizione è sostenuta da diversi fattori, quali anomalie emoreologiche, disfunzioni endoteliali, anomalie piastriniche o delle vie della coagulazione e fibrinolitica. Tra gli altri fattori, un aumento dell’attività piastrinica contribuisce in modo significativo allo stato pro -trombotico. Le piastrine dei pazienti con ipertensione essenziale mostrano diverse anomalie indicative della loro attivazione. A livello morfologico si riscontra il passaggio ad una forma sferica, l’aumento di massa e di volume. A livello biochimico si ha un incremento della quota intracellulare di calcio ed una maggiore espressione di P-selectina sulla membrana. Infine a livello funzionale le piastrine di questi pazienti mostrano una ridotta sensibilità al NO esogeno. Diversi studi hanno valutato gli effetti del trattamento antiipertensivo nell’attivazione piastrinica. In particolare sono stati presi in considerazione gli ACE-inibitori, i Sartani ed i Calcio Antagonisti. I farmaci che agiscono sul sistema renina angiotensina hanno mostrato effetto neutrale o favorevole, mentre per gli altri è stata evidenziata una netta capacità di ridurre l’aggregazione piastrinica. Per quanto riguarda la terapia antiaggregante vera e propria, i farmaci più ampiamente utilizzati sono l’Aspirina e, di più recente introduzione, il Clopidogrel. Il loro utilizzo migliora la prognosi nei pazienti ipertesi ad alto rischio cardiovascolare ed in quelli in cui è presente malattia aterosclerotica clinicamente evidente. Al contrario, l’uso di tale trattamento nei pazienti ipertesi a basso rischio non è stato ancora ben definito ed è meritevole di ulteriori studi clinici. Un rischio cardiovascolare maggiore del 20% a 10 anni, o un’età superiore a 50 anni con un modesto deterioramento della funzione renale sono i due criteri principali, utilizzati per giustificare il trattamento con basse dosi di Aspirina. L’uso del Clopidogrel offre teoricamente dei vantaggi rispetto all’Aspirina nella prevenzione primaria, avendo dimostrato non solo un effetto antitrombotico, ma anche di prevenire la progressione dell’aterosclerosi nei pazienti ipertesi

Neurogenin3 phosphorylation controls reprogramming efficiency of pancreatic ductal organoids into endocrine cells.

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.This work was supported by: the MRC Research Grant MR/K018329/1 and the Rosetrees Trust and Stoneygate Trust (to A.P. and R.A.); the MRC Research Grant MR/L021129/1 and core support from the Wellcome Trust and MRC Cambridge Stem Cell Institute (to A.P., R.A., B.D.S., B.G.); the Wellcome Trust 098357/Z/12/Z (to B.D.S. and R.A.); the Wellcome Trust 097922/Z/11/Z and the Clinical Research Infrastructure Single-cell Facility MR/M008975/1 (to B.G.). S.C. is supported by a GOSH Charity studentship (V6116). P.B. is supported by the UCL Excellence Fellowship Programme, the European Research Council (ERC-Stg-2014 639429), the Rosetrees Trust (A1411 and A1179) and the NIHR BRC at Great Ormond Street Hospital for Children NHS Foundation Trust. M.H. is a Sir Henry Dale fellow and supported by an EU-H2020 grant, LSMF4LIFE, by the Wellcome Trust 104151/Z/14/A and the Royal Societ

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

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

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds

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

Engineering transplantable jejunal mucosal grafts using patient-derived organoids from children with intestinal failure

In a first step toward developing autologous tissue grafts for the treatment of children with intestinal failure, patient-derived jejunal organoids seeded on scaffolds of decellularized human intestinal matrix formed grafts that had jejunal properties and formed luminal structures when transplanted into mice.Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children(1). The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence(2). 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