Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)-Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK-Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell's environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies.

Publisher Correction: Reprogramming normal cells into tumour precursors requires ECM stiffness and oncogene-mediated changes of cell mechanical properties.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics.

Induced pluripotent stem cells (iPSCs) are generated via the expression of the transcription factors OCT4 (also known as POU5F1), SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naive iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here, we report that human naive iPSCs (niPSCs) can be generated directly from fewer than 1,000 primary human somatic cells, without requiring stable genetic manipulation, via the delivery of modified messenger RNAs using microfluidics. Expression of the OSKM factors in combination with NANOG for 12 days generates niPSCs that are free of transgenes, karyotypically normal and display transcriptional, epigenetic and metabolic features indicative of the naive state. Importantly, niPSCs efficiently differentiate into all three germ layers. While niPSCs can be generated at low frequency under conventional conditions, our microfluidics approach enables the robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.

Cross-talk of healthy and impaired human tissues for dissection of disease pathogenesis.

Systemic diseases affect multiple tissues that interact with each other within a network difficult to explore at the body level. However, understanding the interdependences between tissues could be of high relevance for drug target identification, especially at the first stages of disease development. In vitro systems have the advantages of accessibility to measurements and precise controllability of culture conditions, but currently have limitations in mimicking human in vivo systemic tissue response. In this work, we present an in vitro model of cross-talk between an ex vivo culture of adipose tissue from an obese donor and a skeletal muscle in vitro model from a healthy donor. This is relevant to understand type 2 diabetes mellitus pathogenesis, as obesity is one of its main risk factors. The human adipose tissue biopsy was maintained as a three-dimensional culture for 48 h. Its conditioned culture medium was used to stimulate a human skeletal muscle-on-chip, developed by differentiating primary cells of a patient's biopsy under topological cues and molecular self-regulation. This system has been characterized to demonstrate its ability to mimic important features of the normal skeletal muscle response in vivo. We then found that the conditioned medium from a diseased adipose tissue is able to perturb the normal insulin sensitivity of a healthy skeletal muscle, as reported in the early stages of diabetes onset. In perspective, this work represents an important step toward the development of technological platforms that allow to study and dissect the systemic interaction between unhealthy and healthy tissues in vitro. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2766, 2019.

The SWI/SNF complex is a mechanoregulated inhibitor of YAP and TAZ.

Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression1. However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A-SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ2. Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A-SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A-SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A-SWI/SNF, thereby preventing the formation of the ARID1A-SWI/SNF-YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A-SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis.

YAP/TAZ link cell mechanics to Notch signalling to control epidermal stem cell fate.

How the behaviour of somatic stem cells (SCs) is influenced by mechanical signals remains a black-box in cell biology. Here we show that YAP/TAZ regulation by cell shape and rigidity of the extracellular matrix (ECM) dictates a pivotal SC decision: to remain undifferentiated and grow, or to activate a terminal differentiation programme. Notably, mechano-activation of YAP/TAZ promotes epidermal stemness by inhibition of Notch signalling, a key factor for epidermal differentiation. Conversely, YAP/TAZ inhibition by low mechanical forces induces Notch signalling and loss of SC traits. As such, mechano-dependent regulation of YAP/TAZ reflects into mechano-dependent regulation of Notch signalling. Mechanistically, at least in part, this is mediated by YAP/TAZ binding to distant enhancers activating the expression of Delta-like ligands, serving as 'in cis' inhibitors of Notch. Thus YAP/TAZ mechanotransduction integrates with cell-cell communication pathways for fine-grained orchestration of SC decisions.

3D high-resolution two-photon crosslinked hydrogel structures for biological studies.

Hydrogels are widely used as matrices for cell growth due to the their tuneable chemical and physical properties, which mimic the extracellular matrix of natural tissue. The microfabrication of hydrogels into arbitrarily complex 3D structures is becoming essential for numerous biological applications, and in particular for investigating the correlation between cell shape and cell function in a 3D environment. Micrometric and sub-micrometric resolution hydrogel scaffolds are required to deeply investigate molecular mechanisms behind cell-matrix interaction and downstream cellular processes. We report the design and development of high resolution 3D gelatin hydrogel woodpile structures by two-photon crosslinking. Hydrated structures of lateral linewidth down to 0.5µm, lateral and axial resolution down to a few µm are demonstrated. According to the processing parameters, different degrees of polymerization are obtained, resulting in hydrated scaffolds of variable swelling and deformation. The 3D hydrogels are biocompatible and promote cell adhesion and migration. Interestingly, according to the polymerization degree, 3D hydrogel woodpile structures show variable extent of cell adhesion and invasion. Human BJ cell lines show capability of deforming 3D micrometric resolved hydrogel structures. STATEMENT OF SIGNIFICANCE: The design and development of high resolution 3D gelatin hydrogel woodpile structures by two-photon crosslinking is reported. Significantly, topological and mechanical conditions of polymerized gelatin structures were suitable for cell accommodation in the volume of the woodpiles, leading to a cell density per unit area comparable to the bare substrate. The fabricated structures, presenting micrometric features of high resolution, are actively deformed by cells, both in terms of cell invasion within rods and of cell attachment in-between contiguous woodpiles. Possible biological targets for this 3D approach are customized 3D tissue models, or studies of cell adhesion, deformation and migration.

Hydrogel with Orthogonal Reactive Units: 2D and 3D Cross-Linking Modulation.

In this work, an engineered hydrogel system with a 2D and 3D tunable cross-linking degree is presented. A precise chemical design by the introduction of cross-linkable units, having reaction orthogonality, allows to control the network formation both in time and space and to selectively alter the hydrogel physical properties. Hydrogel chemistry has been tailored in order to produce spatially controlled stiffness changes and drive cell morphology through mechanical cues. Elastic modulus rises by more than double after photocross-linking, as shown by atomic force microscopy measurements. Biological response is also analyzed and stiffness-dependent cell spreading and proliferation are verified. Different pattern geometries are successfully realized by UV lithography, allowing 2D cross-linking modulation. Furthermore, 3D mechanical tuning at micro- and submicrometer scale by two-photon polymerization makes this system a biologically relevant matrix to study cell functions and tissue development.

High-efficiency cellular reprogramming with microfluidics.

We report that the efficiency of reprogramming human somatic cells to induced pluripotent stem cells (hiPSCs) can be dramatically improved in a microfluidic environment. Microliter-volume confinement resulted in a 50-fold increase in efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors. In these small volumes, extracellular components of the TGF-ß and other signaling pathways exhibited temporal regulation that appears critical to acquisition of pluripotency. The high quality and purity of the resulting hiPSCs (µ-hiPSCs) allowed direct differentiation into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion.

Functional differentiation of human pluripotent stem cells on a chip.

Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.

Optimal periodic perfusion strategy for robust long-term microfluidic cell culture.

Long-term cell culture in microfluidic devices is an essential prerequisite for "on a chip" biological and physiological based studies. We investigated how medium delivery, from continuous to periodic perfusion, affects long-term cell cultures in a microfluidic platform. Computational simulations suggested that different delivery strategies result in different temporal profiles of accumulation and washing out of endogenous (EnF) and exogenous (ExF) factors, respectively. Thus, cultures exposed to the same overall amount of medium with different temporal profiles were analysed in terms of homogeneity, cell morphology and phenotype. Murine and human cell lines (C2C12 and HFF) and mouse embryonic stem cells (mESC) were cultured in microfluidic channels. An ad hoc experimental setup was developed to perform continuous and periodic medium delivery into the chip, tuning the flow rate, the perfusion time, and the interval of perfusion while using the same amount of medium volume. Periodic medium delivery with a short perfusion pulse ensured cell homogeneity compared to standard cell culture. Conversely, a continuous flow resulted in cell heterogeneity, with abnormal morphology and vesiculation. Only dramatic and unfeasible increasing of perfused medium volume in the continuous configuration could rescue normal cell behaviour. Consistent results were obtained for C2C12 and HFF. In order to extend these results to highly sensitive cells, mESC were cultured for 6 days in the microfluidic channels. Our analysis demonstrates that a periodic medium delivery with fast pulses (with a frequency of 4 times per day) resulted in a homogeneous cell culture in terms of cell viability, colony morphology and maintenance of pluripotency markers. According to experimental observations, the computational model provided a rational description of the perfusion strategies and of how they deeply shape the cell microenvironment in microfluidic cell cultures. These results provide new insight to define optimal strategies for homogeneous and robust long-term cell culture in microfluidic systems, an essential prerequisite for lab on chip cell-based applications.

A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.

Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.

Biosensing with electroconductive biomimetic soft materials.

The development of smart biomaterials able to quantitatively analyse the dynamics of biological systems with high temporal resolution in biomimetic environments is of paramount importance in biophysics, biology and medicine. In this context, we develop a biosensing water-based soft biomaterial with tunable mechanical properties through the generation of an electroconductive nano-element network. As a proof of concept, in order to detect glucose concentration, we fabricate an electroconductive polyacrylamide glucose oxidase (GOx) loaded hydrogel (HY) modified with a small amount of single-walled carbon nanotubes (SWNTs) (up to 0.85 wt%). MicroRaman maps and optical analysis show the nanotube distribution in the samples at different mass fractions. Electrochemical impedance spectra and their fitting with equivalent circuit models reveal electron conduction in the charged hydrogels in addition to ionic conductivity. The effective resulting resistance of the nanostructured network is comparable to that of a gold electrode. These findings were also confirmed by cyclic voltammetry. Interestingly, heterogeneous clustering of SWNTs shows double electric mechanisms and efficiencies. GOx-SWNT doped hydrogels show a linear glucose concentration response in the range between 0.1 mM and 1.6 mM; taken together these results show high detection limits for glucose (down to 15 µM) and a sensitivity of 0.63 µA mM-1. In the perspective of monitoring cell dynamics, hydrogel functionalization allows cell adhesion and long-term cell culture, and atomic force microscopy is used for mapping the doped hydrogel stiffness. Myoblasts, cells sensitive to mechanical substrate properties, show proper differentiation of phenotype in the SWNT-HYs with nominal physiological stiffness.

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study.

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.

Role of YAP/TAZ in mechanotransduction.

Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

High pressure gases: role of dynamic intracellular pH in pasteurization.

Certain dense gases, including CO(2) and N(2)O, are known to deactivate food pathogens safely. Complete deactivation requires disabling intracellular metabolic pathways by extracellular processing that causes membrane disruption, irreversible denaturation of proteins, or extraction of cell contents. At present, neither the precise physical and metabolic mechanisms nor their kinetics behind dense gas pasteurization are known. The mechanisms depend strongly on both the organism and the environment and may combine membrane disruption, membrane permeabilization, and altering pH. Herein we elucidate the mechanisms of dense gas inactivation with the aid of a novel approach for measuring intracellular pH (pH(i)) under high gas pressure. Using a pH-sensitive GFP-variant of S. cerevisiae as the probe, we demonstrate that membrane permeabilization by a non-acidic gas, N(2)O, contributes to inactivation but at a rate that is relatively low compared to CO(2). CO(2) not only permeabilizes the membrane but also brings about a rapid drop in pH(i), leading to greater deactivation. Mechanistic understanding is vital to develop safe and effective dense gas technologies for food treatment. Knowledge of pH(i) is also important in other cellular processes, including enzyme activity, gene transcription, and protein synthesis. The GFP technique has been demonstrated to be versatile even under pressure.