Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures

open10Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability.openScattolini, Valentina; Luni, Camilla; Zambon, Alessandro; Galvanin, Silvia; Gagliano, Onelia; Ciubotaru, Catalin Dacian; Avogaro, Angelo; Mammano, Fabio; Elvassore, Nicola; Fadini, Gian PaoloScattolini, Valentina; Luni, Camilla; Zambon, Alessandro; Galvanin, Silvia; Gagliano, Onelia; Ciubotaru, CATALIN DACIAN; Avogaro, Angelo; Mammano, Fabio; Elvassore, Nicola; Fadini, GIAN PAOL

Engineering a 3D in vitro model of human skeletal muscle at the single fiber scale

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 \u3bcm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions

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

Confidence from uncertainty - A multi-target drug screening method from robust control theory

<p>Abstract</p> <p>Background</p> <p>Robustness is a recognized feature of biological systems that evolved as a defence to environmental variability. Complex diseases such as diabetes, cancer, bacterial and viral infections, exploit the same mechanisms that allow for robust behaviour in healthy conditions to ensure their own continuance. Single drug therapies, while generally potent regulators of their specific protein/gene targets, often fail to counter the robustness of the disease in question. Multi-drug therapies offer a powerful means to restore disrupted biological networks, by targeting the subsystem of interest while preventing the diseased network from reconciling through available, redundant mechanisms. Modelling techniques are needed to manage the high number of combinatorial possibilities arising in multi-drug therapeutic design, and identify synergistic targets that are robust to system uncertainty.</p> <p>Results</p> <p>We present the application of a method from robust control theory, Structured Singular Value or μ- analysis, to identify highly effective multi-drug therapies by using robustness in the face of uncertainty as a new means of target discrimination. We illustrate the method by means of a case study of a negative feedback network motif subject to parametric uncertainty.</p> <p>Conclusions</p> <p>The paper contributes to the development of effective methods for drug screening in the context of network modelling affected by parametric uncertainty. The results have wide applicability for the analysis of different sources of uncertainty like noise experienced in the data, neglected dynamics, or intrinsic biological variability.</p

Development of cell culture technology for the expansion of homogeneous populations of human stem cells

Stem cell-based therapies have been proposed as promising for the maintenance, regeneration, or replacement of malfunctioning tissues, but suffer from limitations mainly due to scarce cell availability and clinical safety concern related to cell quality. Optimization of stem cell expansion process is an engineering challenge, besides a biological issue. Aim of the work presented is to develop the experimental technology and the rational insight to understand and control stem cell expansion in vitro, in terms of both average and distributed properties of the cell population produced. A rational analysis of the main phenomena involved in a cell culture was achieved, underlining the sources of stem cell heterogeneity in both conventional culture systems and stirred bioreactors. From an experimental point of view, two types of bioreactors were designed, developed, and prototyped. The first, a microliter bioreactor array, was designed based on thermoconvective mixing; this experimental setup is particularly convenient for multiparametric optimization of cell culture conditions. The second, a six-well suspension bioreactor with a working volume of 10 ml/well, was designed and fabricated for coarse process optimization or, alternatively, for small-scale stem cell production; an improved setup was developed to perform stem cell cultures under hypoxia conditions. Both devices were advantageously used to culture human cord blood-derived hematopoietic stem cells, which were then characterized according to the currently available biological assays. In order to rationally optimize the stem cell expansion process, a computational model, based on a population balance approach, was developed, that takes into account receptor and receptor-ligand complex distribution in the cell sample. The model fairly describes intrinsic intra- and inter-generational heterogeneity arising from the process of cell division. These findings could give interesting feedback to experimental design and to define the operative conditions for bioreactor cultures, in order to minimize extrinsic and intrinsic heterogeneity, and to make a step towards a clinically safe and reliable human hematopoietic stem cell expansion process.E' stato prospettato l'impiego di cellule staminali per terapie volte al mantenimento, alla rigenerazione o alla sostituzione di tessuti malfunzionanti. Tuttavia non sono ancora state risolte alcune limitazioni legate principalmente alla scarsa disponibilità di cellule staminali e ai problemi di sicurezza clinica connessi alla qualità cellulare. L'ottimizzazione del processo di espansione cellulare è un sfida ingegneristica, oltre che biologica. Scopo di questa tesi è lo sviluppo di una tecnologia sperimentale e di un quadro razionale che consenta di comprendere e controllare l'espansione di cellule staminali in vitro, sia considerando le proprietà medie che la loro distribuzione nella popolazione cellulare prodotta. E' stata realizzata un'analisi razionale dei principali fenomeni coinvolti nella coltura cellulare, ponendo in evidenza le fonti di eterogeneità sia nei sistemi di coltura convenzionali che nei bioreattori mescolati. Da un punto di vista sperimentale, sono stati progettati e sviluppati due tipi di bioreattori fino a realizzarne dei prototipi. Il primo, un sistema di bioreattori di volume dell'ordine dei microlitri, è stato progettato basato su un meccanismo di termoconvezione; questo apparato sperimentale è particolarmente adatto per un'ottimizzazione multiparametrica delle condizioni di coltura. Il secondo, un bioreattore in sospensione multipozzetto con un volume operativo di 10 ml/pozzetto, è stato pensato e costruito per un'ottimizzazione di processo meno dettagliata o, alternativamente, per una produzione su piccola scala di cellule staminali; una versione più avanzata è stata sviluppata per effettuare colture di cellule staminali in condizioni di ipossia. Entrambi i dispositivi sono stati vantaggiosamente utilizzati per coltivare cellule staminali ematopoietiche, ricavate da cordone ombelicale umano, che sono poi state caratterizzate secondo i metodi di analisi biologica convenzionali. Per ottimizzare razionalmente il processo di espansione delle cellule staminali, è stato sviluppato un modello computazionale, basato su un bilancio di popolazione, che tiene conto della distribuzione di recettori e di complessi recettore-ligando nel campione cellulare. Il modello descrive ragionevolmente l'eterogeneità intrinseca, intra- e intergenerazionale, derivante dal processo di divisione cellulare. Questi risultati possono dare un riscontro positivo in fase di progettazione degli esperimenti e di definizione delle condizioni operative a cui effettuare colture in bioreattore, al fine di minimizzare l'eterogeneità estrinseca e intrinseca della popolazione cellulare e di effettuare un ulteriore avanzamento verso un processo di espansione di cellule staminali umane clinicamente sicuro ed affidabile

Derivation and Differentiation of Human Pluripotent Stem Cells in Microfluidic Devices

An integrative approach based on microfluidic design and stem cell biology enables capture of the spatial-temporal environmental evolution underpinning epigenetic remodeling and the morphogenetic process. We examine the body of literature that encompasses microfluidic applications where human induced pluripotent stem cells are derived starting from human somatic cells and where human pluripotent stem cells are differentiated into different cell types. We focus on recent studies where the intrinsic features of microfluidics have been exploited to control the reprogramming and differentiation trajectory at the microscale, including the capability of manipulating the fluid velocity field, mass transport regime, and controllable composition within micro- to nanoliter volumes in space and time. We also discuss studies of emerging microfluidic technologies and applications. Finally, we critically discuss perspectives and challenges in the field and how these could be instrumental for bringing about significant biological advances in the field of stem cell engineering

Robust multi-drug therapy design and application to insulin resistance in type 2 diabetes

Single-drug therapies often fail in the treatment of complex diseases, such as diabetes, because they act on a specific target, and are often by-passed by the redundant mechanisms that confer robustness to biological systems, both in health and in disease. On the other hand, multi-drug therapies (MDTs) show greater efficacy, but efficient methodologies for their design are needed to handle the high number of combinatorial possibilities. In this paper, we describe a methodology for MDT design based on the structured singular value, a tool from the robust control theory. The procedure can be applied to a mathematical model of the biological system under consideration, and it includes medical performance robustness as a design requirement. This aspect is of primary importance because robustness has been recognized as an intrinsic property of biological systems that maintain performance despite a variety of environmental conditions and the subject-to-subject variability of response. Furthermore, the development of a mathematical model always includes a certain level of uncertainty, due to the inevitable assumptions involved in its development, and the often scarce availability of experimental data. The methodology is here applied to the insulin resistance condition, a primary impairment in the development of type 2 diabetes. Results from simulations confirm that the performance and robustness objectives are achieved under different input dynamics, and suggest potentially powerful therapeutic vector targets

Human-on-chip for therapy development and fundamental science

Organ-on-chip systems integrate microfluidic technology and living cells to study human physiology and pathophysiology. These human in vitro models are promising substitutes for animal testing, and their small scale enables precise control of culture conditions and high-throughput experiments, which would not be economically sustainable on a macroscopic level. Multiple sources of biological material are used in the development of organ-on-chips, from biopsies to stem cells. Each source has its own peculiarities and technical requirements for integration into microfluidic chips, and is suitable for specific applications. While a biopsy is the tissue of choice for the biomimetic response to ageing, induced pluripotent stem cells hold great promise for the study of genetic-related disease pathogenesis, and primary cultures can fill the gap