Human gastro-intestinal organoid engineering: a state of the art

Gastrointestinal organ failure, from congenital or postnatally acquired pathologies, is a major cause of death across countries of all income levels. Organoids and engineered tissues have been widely investigated as tools to model organ functions and treat pathologies. In this review we aim to describe the progress in human organoid engineering applied to the gastrointestinal tract (namely esophagus, stomach, and intestine). Starting from the onset of the organoid culture technique, we illustrate genetic engineering, stem cell niche engineering, bioprinting, and microfluidics approaches used to integrate mechano-physiological parameters with human organoids. Thanks to these improvements, organoid technology allows disease modelling of patient-specific pathologies, and personalized treatment screening, also offering a cell source for autologous transplantation. We further present an overview of the advances of tissue engineering in animal systems, concerning novel materials and scaffolds to be combined with a variety of cell types to reconstitute a viable surrogate for implantation. The effort in this field sets organoids as an important tool in personalized and regenerative medicine. Their application combined with the advances in tissue engineering holds great potential for translational application

Inflammatory cytokines and VEGF measured in exhaled breath condensate are correlated with tumor mass in non-small cell lung cancer.

Inflammation mediated by the immune system is known to be important in carcinogenesis and, specifically, T helper 17 cells have been reported to play a role in tumor progression by promoting neo-angiogenesis. The aim of this study was to investigate whether inflammatory cytokines and vascular endothelial growth factor (VEGF) levels in exhaled breath condensate (EBC) and in serum were related to tumor size in patients with non-small cell lung cancer (NSCLC). Il-6, IL-17, TNF-α and VEGF levels were measured in EBC and serum of 15 patients with stage I-IIA NSCLC and in 30 healthy controls by immunoassay. The tumor size was measured by a CT scan. The concentrations of IL-6, IL-17 and VEGF were significantly higher in EBC of patients with lung cancer, compared with controls, while only serum IL-6 concentration was higher in patients compared to controls. A significant correlation (r = 0.78, p = 0.001) was observed between EBC levels of IL-6 and IL-17; IL-17 was also correlated to EBC levels of the VEGF (r = 0.83, p < 0.001) and TNF-α (r = 0.62, p = 0.014). The tumor diameter was significantly correlated with EBC concentrations of VEGF (r = 0.58, p = 0.039), IL-6 (r = 0.67, p = 0.013) and IL-17 (r = 0.66, p = 0.017). Our results show a significant relationship between inflammatory and angiogenic markers, measured in EBC by a non-invasive method, and tumor mass

Long-term maintenance of dried acellular matrices

[EN] Dried and sterile acellular esophageal matrix was obtained within a new drying process based on the use of supercritical carbon dioxide (SC-CO2). Experiments were performed coupling a conventional detergent enzymatic treatment with two different drying methods: (i) SC-CO2 drying alone; (ii) dehydration in ethanol and a subsequent SC-CO2 drying. Long term preservation was achieved for several months after drying, demonstrating the maintenance of extracellular matrix (ECM) structure, mechanical properties and biocompatibility within cell repopulation studies in vitro. Overall, the results highlighted the potential of this novel technology to obtain a dry and sterile acellular matrix that can be easily stored for oesophageal regeneration in patients with emergency need.The research leading to these results received funding from Cassa di Risparmio di Trento e Rovereto (CaRiTRo) within the research project "Supercritical decellularization of engineered tissues for clinical application", biomedical science section, 2013. We thanks Lorenza Lazzari for the donation of BM-MSCs from the Cell Factory Bank (Milan-Italy).Zambon, A.; Giobbe, G.; Vetralla, M.; Michelino, F.; Urbani, L.; Pantano, M.; Pugno, N.... (2018). Long-term maintenance of dried acellular matrices. En IDS 2018. 21st International Drying Symposium Proceedings. Editorial Universitat Politècnica de València. 1091-1097. https://doi.org/10.4995/IDS2018.2018.7844OCS1091109

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.Several clinical reports have described gastrointestinal symptoms for COVID-19, though whether the virus can replicate within the stomach remains unclear. Here the authors generate gastric organoids from human biopsies and show that the virus can efficiently infect gastric epithelium, suggesting that the stomach might have an active role in fecal-oral transmission

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy

Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins

Microtechnology-aided differentiation of human pluripotent stem cells into hepatocyte-like cells

There is a growing interest by scientific community, together with pharmaceutical companies and clinical researcher on finding valid alternatives to standard models in the development of new therapeutic strategies and robust drug screening processes. Research still relies on the use of immortalized cell lines, primary cell extracted from living organs and animal models. These methodologies are valid and still mainstream for general purposes, but most of the times present evident limits. Cell lines and primary cells often fail to reproduce the exact characteristics of the tissue found in vivo, because they lose some of the features and functionalities when grown in culture. On the other hand, animal models, even if necessary for the study of specific diseases and new compound testing, are expensive and time-consuming, and often show poor predictive capacity and scarce reproducibility of the human condition. Hence, human pluripotent stem cells could represent a valid alternative to the existing models, thank to their capacity to be expanded indefinitely and differentiate into almost all cell types found in vivo. In the recent years, there has been a growing attention on engineered tissues differentiate from pluripotent cells. They have the capacity to transform the way to study human pathophysiology and physiology in vitro. Nevertheless, there are still major problems in the process of differentiating human embryonic and induced pluripotent stem cell in vitro. This is because of the difficulty to specifically direct cell fate to a particular cell type in a robust way, and for the poor reproduction of the physiological conditions under which these processes take place. In this direction, new microtechnologies could help overcome these major limitations, because they allow working on microscales, in a way that cannot be reproduced by standard culture conditions. In this context, the aim of this PhD thesis is to efficiently differentiate human pluripotent stem cells in order to obtain functional relevant cell types, such as cardiomyocytes and hepatocytes. The strategy applied for the obtainment of these human in vitro models rely on the application of microscale technologies for reproducing in vitro the main physiological cues, which guide differentiation and allow functional development. In particular, three-dimensional microwell were used for modulating endogenous factor accumulation on differentiating human embryoid bodies, to screen for selective germ layer commitment guided by a differential microenvironment around the differentiating cells. Furthermore, a mechanical modulation of the pluripotent nuclei mechanical properties was imposed with the use of microstructured substrate, for the study of the peculiar capacity of nuclear cell deformation, and its effect in pluripotency and early differentiation. Moreover, microfluidic technologies were used to selectively modulate cell soluble microenvironment, in order to optimize pluripotency maintenance, early germ layer commitment, and functional differentiation into cardiomyocytes and hepatocytes. A high percentage of spontaneously beating cardiomyocytes on chip was obtained, showing proper functional response to calcium stimuli. On the other hand, microfluidic technology allowed to obtain a higher percentage of hepatocytes compared to standard culture conditions. These cells showed proper phenotypic and functional characteristics, which were also analyzed in a more physiological condition under a defined oxygen gradient, mimicking in vivo physiological conditions. These specific cell types generated on chip from human pluripotent stem cells through a multi-stage approach show specific functional differentiation, which opens a new perspective for multi-parametric and large scale human tissue-based screening assays.Vi è un crescente interesse fra la comunità scientifica internazionale, le compagnie farmaceutiche e la ricerca clinica nel trovare delle valide alternative ai modelli standard nello sviluppo di nuove strategie terapeutiche e di processi di scoperta di nuovi farmaci. I ricercatori si basano tuttora sull’utilizzo di linee cellulari immortalizzate, cellule primarie estratte da organi e su modelli animali. Queste metodiche sono valide e largamente utilizzate per scopi generali, ma il più delle volte presentano dei limiti evidenti. Le linee cellulari e cellule primarie spesso non riproducono fedelmente le esatte caratteristiche dei tessuti in vivo, poiché perdono alcune caratteristiche fisiche e funzionali quando sono coltivate in vitro. D’altra parte, i modelli animali sono ancora necessari per lo studio di patologie specifiche e nel test di nuovi composti, ma risultano essere dispendiosi in termini di tempo e denaro e spesso mostrano una scarsa capacità predittiva nei confronti degli effetti sull’uomo. Quindi, le cellule staminali pluripotenti umane rappresentano una valida alternativa ai modelli esistenti, grazie alle loro capacità di essere espanse in vitro indefinitamente e di poter differenziare in tutti i tipi cellulari derivanti dai tre foglietti germinali. Negli ultimi anni l’attenzione si è concentrata sui tessuti ingegnerizzati, differenziati a partire da cellule pluripotenti. Questi hanno la possibilità di trasformare radicalmente il modo in cui studiamo in vitro la fisiologia e la patofisiologia umana. Ciò non di meno, vi sono ancora problemi nei processi di differenziamento di cellule staminali embrionali e pluripotenti indotte umane in vitro. Questo perché si riscontra difficoltà nel dirigere specificamente il destino cellulare verso un determinato tipo cellulare in modo robusto, e per la scarsa riproducibilità delle condizioni fisiologiche in cui questi processi hanno normalmente luogo in vivo. In questo ambito, le nuove micro-tecnologie possono dare un aiuto nell’oltrepassare queste limitazioni, poiché permettono di lavorare in micro-scala in maniera difficilmente riproducibile in condizioni di coltura standard. In questo contesto, lo scopo di questa tesi di dottorato è quello di differenziare efficacemente cellule staminali pluripotenti umane per riuscire ad ottenere rilevanti tipi cellulari, quali cardiomiociti ed epatociti. La strategia applicata per l’ottenimento di questi modelli umani in vitro si basa sull’applicazione di tecnologie in micro-scala per permettere la riproduzione in vitro delle nicchie fisiologiche, che guidano il differenziamento e permettono lo sviluppo funzionale. In particolare sono stati utilizzati dei micro-pozzetti tridimensionali per modulare l’accumulo di fattori endogeni in corpi embrioidi umani in differenziamento, per studiare lo sviluppo dei tre foglietti germinali guidato da diversi microambienti cellulari. È stata poi imposta una modulazione delle proprietà meccaniche dei nuclei di cellule pluripotenti tramite utilizzo di substrati micro-strutturati, per lo studio della capacità peculiare di deformazione nucleare di tali cellule e si è valutato l’effetto sulla pluripotenza e il differenziamento precoce. Inoltre sono state utilizzate tecnologie micro-fluidiche per modulare selettivamente il microambiente cellulare solubile, in modo da ottimizzare il mantenimento della pluripotenza in chip micro-fluidici, nonché lo sviluppo cellulare precoce e il differenziamento funzionale in cardiomiociti ed epatociti. È stata ottenuta un’alta percentuale di cardiomiociti contrattili nei chip che mostravano risposte funzionali corrette a stimoli di calcio. La tecnologia micro-fluidica ha permesso poi di ottenere un’alta percentuale di epatociti in chip rispetto alle condizioni di coltura standard. Queste cellule mostravano caratteristiche fenotipiche e funzionali corrette, che sono state poi analizzate in condizioni più fisiologiche sotto un gradiente stabile di ossigeno, mimando le condizioni in vivo. Questi tipi cellulari specifici, generati in chip da cellule staminali pluripotenti umane tramite un approccio multi-stadio, mostrano un differenziamento funzionale specifico, che apre a nuove prospettive per test multi-parametrici su larga scala basati su tessuti funzionali umani