Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilization of Bioemulsions Enabling Adherent Cell Expansion.

Bioemulsions are attractive platforms for the scalable expansion of adherent cells and stem cells. In these systems, cell adhesion is enabled by the assembly of protein nanosheets that display high interfacial shear moduli and elasticity. However, to date, most successful systems reported to support cell adhesion at liquid substrates have been based on coassemblies of protein and reactive cosurfactants, which limit the translation of bioemulsions. In this report, we describe the design of protein nanosheets based on two globular proteins, bovine serum albumin (BSA) and ß-lactoglobulin (BLG), biofunctionalized with RGDSP peptides to enable cell adhesion. The interfacial mechanics of BSA and BLG assemblies at fluorinated liquid-water interfaces is studied by interfacial shear rheology, with and without cosurfactant acyl chloride. Conformational changes associated with globular protein assembly are studied by circular dichroism and protein densities at fluorinated interfaces are evaluated via surface plasmon resonance. Biofunctionalization mediated by sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) is studied by fluorescence microscopy. On the basis of the relatively high elasticities observed in the case of BLG nanosheets, even in the absence of cosurfactant, the adhesion and proliferation of mesenchymal stem cells and human embryonic kidney (HEK) cells on bioemulsions stabilized by RGD-functionalized protein nanosheets is studied. To account for the high cell spreading and proliferation observed at these interfaces, despite initial moderate interfacial elasticities, the deposition of fibronectin fibers at the surface of corresponding microdroplets is characterized by immunostaining and confocal microscopy. These results demonstrate the feasibility of achieving high cell proliferation on bioemulsions with protein nanosheets assembled without cosurfactants and establish strategies for rational design of scaffolding proteins enabling the stabilization of interfaces with strong shear mechanics and elasticity, as well as bioactive and cell adhesive properties. Such protein nanosheets and bioemulsions are proposed to enable the development of new generations of bioreactors for the scale up of cell manufacturing.

Mesenchymal Stem Cells Sense the Toughness of Nanomaterials and Interfaces.

Stem cells are known to sense and respond to the mechanical properties of biomaterials. In turn, cells exert forces on their environment that can lead to striking changes in shape, size and contraction of associated tissues, and may result in mechanical disruption and functional failure. However, no study has so far correlated stem cell phenotype and biomaterials toughness. Indeed, disentangling toughness-mediated cell response from other mechanosensing processes has remained elusive as it is particularly challenging to uncouple Youngs' or shear moduli from toughness, within a range relevant to cell-generated forces. In this report, it is shown how the design of the macromolecular architecture of polymer nanosheets regulates interfacial toughness, independently of interfacial shear storage modulus, and how this controls the expansion of mesenchymal stem cells at liquid interfaces. The viscoelasticity and toughness of poly(l-lysine) nanosheets assembled at liquid-liquid interfaces is characterised via interfacial shear rheology. The local (microscale) mechanics of nanosheets are characterised via magnetic tweezer-assisted interfacial microrheology and the thickness of these assemblies is determined from in situ ellipsometry. Finally, the response of mesenchymal stem cells to adhesion and culture at corresponding interfaces is investigated via immunostaining and confocal microscopy.

Impact of the multiscale viscoelasticity of quasi-2D self-assembled protein networks on stem cell expansion at liquid interfaces.

Although not typically thought to sustain cell adhesion and expansion, liquid substrates have recently been shown to support such phenotypes, providing protein nanosheets could be assembled at corresponding liquid-liquid interfaces. However, the precise mechanical properties required from such quasi-2D nanoassemblies and how these correlate with molecular structure and nanoscale architecture has remained unclear. In this report, we screen a broad range of surfactants, proteins, oils and cell types and correlate interfacial mechanical properties with stem cell expansion. Correlations suggest an impact of interfacial viscoelasticity on the regulation of such behaviour. We combine interfacial rheology and magnetic tweezer-based interfacial microrheology to characterise the viscoelastic profile of protein nanosheets assembled at liquid-liquid interfaces. Based on neutron reflectometry and transmission electron microscopy data, we propose that the amorphous nanoarchitecture of quasi-2D protein nanosheets controls their multi-scale viscoelasticity which, in turn, correlates with cell expansion. This understanding paves the way for the rational design of protein nanosheets for microdroplet and bioemulsion-based stem cell manufacturing and screening platforms.

Adaptive Lipid Immiscibility and Membrane Remodeling Are Active Functional Determinants of Primary Ciliogenesis.

Lipid liquid-liquid immiscibility and its consequent lateral heterogeneity have been observed under thermodynamic equilibrium in model and native membranes. However, cholesterol-rich membrane domains, sometimes referred to as lipid rafts, are difficult to observe spatiotemporally in live cells. Despite their importance in many biological processes, robust evidence for their existence remains elusive. This is mainly due to the difficulty in simultaneously determining their chemical composition and physicochemical nature, whilst spatiotemporally resolving their nanodomain lifetime and molecular dynamics. In this study, a bespoke method based on super-resolution stimulated emission depletion (STED) microscopy and raster imaging correlation spectroscopy (RICS) is used to overcome this issue. This methodology, laser interleaved confocal RICS and STED-RICS (LICSR), enables simultaneous tracking of lipid lateral packing and dynamics at the nanoscale. Previous work indicated that, in polarized epithelial cells, the midbody remnant licenses primary cilium formation through an unidentified mechanism. LICSR shows that lipid immiscibility and its adaptive collective nanoscale self-assembly are crucial for the midbody remnant to supply condensed membranes to the centrosome for the biogenesis of the ciliary membrane. Hence, this work poses a breakthrough in the field of lipid biology by providing compelling evidence of a functional role for liquid ordered-like membranes in primary ciliogenesis.

Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion.

The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant-associated membrane patch (RAMP), once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of the spatiotemporal lipid collective assembly at the plasma membrane as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.

Methods to Generate Tube Micropatterns for Epithelial Morphogenetic Analyses and Tissue Engineering.

Cells live in a highly curved and folded 3D microenvironment within the human body. Since epithelial cells in internal organs usually adopt a tubular shape, there is a need to engineer simple in vitro devices to promote this cellular configuration. The aim of these devices would be to investigate epithelial morphogenesis and cell behavior-leading to the development of more sophisticated platforms for tissue engineering and regenerative medicine. In this chapter, we first explain the need for such epithelial tubular micropatterns based on anatomical considerations and then survey methods that can be used to study different aspects of epithelial tubulogenesis. The methods examined can broadly be divided into two classes: conventional 2D microfabrication for the formation of simple epithelial tubes in substrates of different stiffness; and 3D approaches to enable the self-assembly of organoid-derived epithelial tubes in a tubular configuration. These methods demonstrate that modeling tubulogenesis in vitro with high resolution, accuracy, and reproducibility is possible.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis.

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

Micropattern-based platform as a physiologically relevant model to study epithelial morphogenesis and nephrotoxicity.

Tubulogenesis in epithelial organs often initiates with the acquisition of apicobasal polarity, giving rise to the formation of small lumens that expand and fuse to generate a single opened cavity. In this study, we present a micropattern-based device engineered to generate epithelial tubes through a process that recapitulates in vivo tubule morphogenesis. Interestingly, tubulogenesis in this device is dependent on microenvironmental cues such as cell confinement, extracellular matrix composition, and substrate stiffness, and our set-up specifically allows the control of these extracellular conditions. Additionally, proximal tubule cell lines growing on micropatterns express higher levels of drug transporters and are more sensitive to nephrotoxicity. These tubes display specific morphological defects that can be linked to nephrotoxicity, which would be helpful to predict potential toxicity when developing new compounds. This device, with the ability to recapitulate tube formation in vitro, has emerged as a powerful tool to study the molecular mechanisms involved in organogenesis and, by being more physiologically relevant than existing cellular models, becomes an innovative platform to conduct drug discovery assays.

Sfrp3 modulates stromal-epithelial crosstalk during mammary gland development by regulating Wnt levels.

Mammary stroma is essential for epithelial morphogenesis and development. Indeed, postnatal mammary gland (MG) development is controlled locally by the repetitive and bi-directional cross-talk between the epithelial and the stromal compartment. However, the signalling pathways involved in stromal-epithelial communication are not entirely understood. Here, we identify Sfrp3 as a mediator of the stromal-epithelial communication that is required for normal mouse MG development. Using Drosophila wing imaginal disc, we demonstrate that Sfrp3 functions as an extracellular transporter of Wnts that facilitates their diffusion, and thus, their levels in the boundaries of different compartments. Indeed, loss of Sfrp3 in mice leads to an increase of ductal invasion and branching mirroring an early pregnancy state. Finally, we observe that loss of Sfrp3 predisposes for invasive breast cancer. Altogether, our study shows that Sfrp3 controls MG morphogenesis by modulating the stromal-epithelial cross-talk during pubertal development.

Mechanosensitive adhesion complexes in epithelial architecture and cancer onset.

Mechanical signals from the extracellular space are paramount to coordinate tissue morphogenesis and homeostasis. Although there is a wide variety of cellular mechanisms involved in transducing extracellular forces, recent literature emphasizes the central role of two main adhesion complexes in epithelial mechanosensitive processes: focal adhesions and adherens junctions. These biomechanical sensors can decode physical signals such as matrix stiffness or intercellular tension into a wide range of coordinated cellular responses, which can impact cell differentiation, migration, and proliferation. Communication between cells and their microenvironment plays a pivotal role both in physiological and pathological conditions. Here we summarize the most recent findings on the biology of these mechanotransduction pathways in epithelial cells, highlighting the extensive amount of biological processes coordinated by cell-matrix and cell-cell adhesion complexes.

Developmental regulation of apical endocytosis controls epithelial patterning in vertebrate tubular organs.

Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.

Genomic instability of surgical sample and cancer-initiating cell lines from human glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in the adult human, with an average survival of 16 months. A small population of cells within the GBM termed cancer-initiating cells is responsible for the initiation and maintenance of the tumor mass. The traditional glioblastoma cancer cells, grown with serum containing media, display increased rate of genomic instability events, which in turn renders the cell cultures with little resembling to the original tumor, making doubtful their use as preclinical models for screening therapeutic agents. On the contrary, the cancer-initiating cells grown in serum-free media seems to show lower rate of genomic instability processes. However, considering the diversity of genetic and/or epigenetic background, we will need to evaluate the possibility of using different culture conditions to allow for the isolation and culture of such cancer-initiating cells diversity, keeping, at the same time, the genomic instability rate as the original tumor. We summarized the main genetic and epigenetic mechanisms that are driving genomic instability in cancer-initiating cells from human glioblastoma.