Organoids and organ-on-chip technology for investigating host-microorganism interactions.

Recent advances in organoid and organ-on-chip (OoC) technologies offer an unprecedented level of tissue mimicry. These models can recapitulate the diversity of cellular composition, 3D organization, and mechanical stimulation. These approaches are intensively used to understand complex diseases. This review focuses on the latest advances in this field to study host-microorganism interactions.

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria.

Synthetic circuit design is crucial for engineering microbes that process environmental cues and provide biologically relevant outputs. To reliably scale-up circuit complexity, the availability of parts toolkits is central. Streptococcus pyogenes (sp)-derived CRISPR interference/dead-Cas9 (CRISPRi/spdCas9) is widely adopted for implementing programmable regulations in synthetic circuits, and alternative CRISPRi systems will further expand our toolkits of orthogonal components. Here, we showcase the potential of CRISPRi using the engineered dCas9 from Staphylococcus aureus (sadCas9), not previously used in bacterial circuits, that is attractive for its low size and high specificity. We designed a collection of ∼20 increasingly complex circuits and variants in Escherichia coli, including circuits with static function like one-/two-input logic gates (NOT, NAND), circuits with dynamic behavior like incoherent feedforward loops (iFFLs), and applied sadCas9 to fix a T7 polymerase-based cascade. Data demonstrated specific and efficient target repression (100-fold) and qualitatively successful functioning for all circuits. Other advantageous features included low sadCas9-borne cell load and orthogonality with spdCas9. However, different circuit variants showed quantitatively unexpected and previously unreported steady-state responses: the dynamic range, switch point, and slope of NOT/NAND gates changed for different output promoters, and a multiphasic behavior was observed in iFFLs, differing from the expected bell-shaped or sigmoidal curves. Model analysis explained the observed curves by complex interplays among components, due to reporter gene-borne cell load and regulator competition. Overall, CRISPRi/sadCas9 successfully expanded the available toolkit for bacterial engineering. Analysis of our circuit collection depicted the impact of generally neglected effects modulating the shape of component dose-response curves, to avoid drawing wrong conclusions on circuit functioning.

Spatial confinement of Trypanosoma brucei in microfluidic traps provides a new tool to study free swimming parasites.

Trypanosoma brucei is the causative agent of African trypanosomiasis and is transmitted by the tsetse fly (Glossina spp.). All stages of this extracellular parasite possess a single flagellum that is attached to the cell body and confers a high degree of motility. While several stages are amenable to culture in vitro, longitudinal high-resolution imaging of free-swimming parasites has been challenging, mostly due to the rapid flagellar beating that constantly twists the cell body. Here, using microfabrication, we generated various microfluidic devices with traps of different geometrical properties. Investigation of trap topology allowed us to define the one most suitable for single T. brucei confinement within the field of view of an inverted microscope while allowing the parasite to remain motile. Chips populated with V-shaped traps allowed us to investigate various phenomena in cultured procyclic stage wild-type parasites, and to compare them with parasites whose motility was altered upon knockdown of a paraflagellar rod component. Among the properties that we investigated were trap invasion, parasite motility, and the visualization of organelles labelled with fluorescent dyes. We envisage that this tool we have named "Tryp-Chip" will be a useful tool for the scientific community, as it could allow high-throughput, high-temporal and high-spatial resolution imaging of free-swimming T. brucei parasites.

SCRIB controls apical contractility during epithelial differentiation.

Although mutations in the SCRIB gene lead to multiple morphological organ defects in vertebrates, the molecular pathway linking SCRIB to organ shape anomalies remains elusive. Here, we study the impact of SCRIB-targeted gene mutations during the formation of the gut epithelium in an organ-on-chip model. We show that SCRIB KO gut-like epithelia are flatter with reduced exposed surface area. Cell differentiation on filters further shows that SCRIB plays a critical role in the control of apical cell shape, as well as in the basoapical polarization of myosin light chain localization and activity. Finally, we show that SCRIB serves as a molecular scaffold for SHROOM2/4 and ROCK1 and identify an evolutionary conserved SHROOM binding site in the SCRIB carboxy-terminal that is required for SCRIB function in the control of apical cell shape. Our results demonstrate that SCRIB plays a key role in epithelial morphogenesis by controlling the epithelial apical contractility during cell differentiation.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

4D live imaging and computational modeling of a functional gut-on-a-chip evaluate how peristalsis facilitates enteric pathogen invasion.

Physical forces are essential to biological function, but their impact at the tissue level is not fully understood. The gut is under continuous mechanical stress because of peristalsis. To assess the influence of mechanical cues on enteropathogen invasion, we combine computational imaging with a mechanically active gut-on-a-chip. After infecting the device with either of two microbes, we image their behavior in real time while mapping the mechanical stress within the tissue. This is achieved by reconstructing three-dimensional videos of the ongoing invasion and leveraging on-manifold inverse problems together with viscoelastic rheology. Our results show that peristalsis accelerates the destruction and invasion of intestinal tissue by Entamoeba histolytica and colonization by Shigella flexneri. Local tension facilitates parasite penetration and activates virulence genes in the bacteria. Overall, our work highlights the fundamental role of physical cues during host-pathogen interactions and introduces a framework that opens the door to study mechanobiology on deformable tissues.

Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1.

The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell-cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome-microtubule network.

SARS-CoV-2 infection induces the dedifferentiation of multiciliated cells and impairs mucociliary clearance.

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Microfabricated Device for High-Resolution Imaging of Preimplantation Embryos.

The mouse preimplantation embryo is an excellent system for studying how mammalian cells organize dynamically into increasingly complex structures. Accessible to experimental and genetic manipulations, its normal or perturbed development can be scrutinized ex vivo by real-time imaging from fertilization to late blastocyst stage. High-resolution imaging of multiple embryos at the same time can be compromised by embryos displacement during imaging. We have developed an inexpensive and easy-to-produce imaging device that facilitates greatly the imaging of preimplantation embryo. In this chapter, we describe the different steps of production and storage of the imaging device as well as its use for live imaging of mouse preimplantation embryos expressing fluorescent reporters from genetically modified alleles or after in vitro transcribed mRNA transfer by microinjection or electroporation.

Bioengineered Human Organ-on-Chip Reveals Intestinal Microenvironment and Mechanical Forces Impacting Shigella Infection.

Intestinal epithelial cells are constantly exposed to pathogens and mechanical forces. However, the impact of mechanical forces on infections leading to diarrheal diseases remains largely unknown. Here, we addressed whether flow and peristalsis impact the infectivity of the human pathogen Shigella within a 3D colonic epithelium using Intestine-Chip technology. Strikingly, infection is significantly increased and minimal bacterial loads are sufficient to invade enterocytes from the apical side and trigger loss of barrier integrity, thereby shifting the paradigm about early stage Shigella invasion. Shigella quickly colonizes epithelial crypt-like invaginations and demonstrates the essential role of the microenvironment. Furthermore, by modulating the mechanical forces of the microenvironment, we find that peristalsis impacts Shigella invasion. Collectively, our results reveal that Shigella leverages the intestinal microenvironment by taking advantage of the microarchitecture and mechanical forces to efficiently invade the intestine. This approach will enable molecular and mechanistic interrogation of human-restricted enteric pathogens.

Biophysical Control of Bile Duct Epithelial Morphogenesis in Natural and Synthetic Scaffolds.

The integration of bile duct epithelial cells (cholangiocytes) in artificial liver culture systems is important in order to generate more physiologically relevant liver models. Understanding the role of the cellular microenvironment on differentiation, physiology, and organogenesis of cholangiocytes into functional biliary tubes is essential for the development of new liver therapies, notably in the field of cholangiophaties. In this study, we investigated the role of natural or synthetic scaffolds on cholangiocytes cyst growth, lumen formation and polarization. We demonstrated that cholangiocyte cyst formation efficiency can be similar between natural and synthetic matrices provided that the mechanical properties of the hydrogels are matched. When using synthetic matrices, we also tried to understand the impact of elasticity, matrix metalloprotease-mediated degradation and integrin ligand density on cyst morphogenesis. We demonstrated that hydrogel stiffness regulates cyst formation. We found that controlling integrin ligand density was key in the establishment of large polarized cysts of cholangiocytes. The mechanism of lumen formation was found to rely on cell self-organization and proliferation. The formed cholangiocyte organoids showed a good MDR1 (multi drug resistance protein) transport activity. Our study highlights the advantages of fully synthetic scaffold as a tool to develop bile duct models.

Artificial niche microarrays for identifying extrinsic cell-fate determinants.

The complex cellular microenvironment plays an important role in determining cell fate. For example, stem cells located in a microenvironment termed niche integrate a wide variety of extrinsic cues to take distinct fate choices. Capturing this multiple-input/multiple-output system in vitro has proven to be very challenging. In order to address this issue, we developed and validated a microfabricated cellular array platform, termed artificial niche microarrays, which is capable of performing high-throughput single-cell assays under physiologically relevant conditions. The platform allows exposing cultured cells to differential signaling cues displayed on soft hydrogel substrates having variable stiffness. The behavior of the seeded cells can be readily quantified across over 2000 multivariate microenvironments. Here we describe a pipeline for performing multifactorial, image-based assays with these artificial niche microarrays. The procedure details the steps from microarray production, cell culture, cell phenotyping, data extraction to statistical analysis.

Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells.

The in vitro expansion of long-term hematopoietic stem cells (HSCs) remains a substantial challenge, largely because of our limited understanding of the mechanisms that control HSC fate choices. Using single-cell multigene expression analysis and time-lapse microscopy, here we define gene expression signatures and cell cycle hallmarks of murine HSCs and the earliest multipotent progenitors (MPPs), and analyze systematically single HSC fate choices in culture. Our analysis revealed twelve differentially expressed genes marking the quiescent HSC state, including four genes encoding cell-cell interaction signals in the niche. Under basal culture conditions, most HSCs rapidly commit to become early MPPs. In contrast, when we present ligands of the identified niche components such as JamC or Esam within artificial niches, HSC cycling is reduced and long-term multipotency in vivo is maintained. Our approach to bioengineer artificial niches should be useful in other stem cell systems.Haematopoietic stem cell (HSC) self-renewal is not sufficiently understood to recapitulate in vitro. Here, the authors generate gene signature and cell cycle hallmarks of single murine HSCs, and use identified endothelial receptors Esam and JamC as substrates to enhance HSC growth in engineered niches.

Nitric Oxide Releasing Coronary Stent: A New Approach Using Layer-by-Layer Coating and Liposomal Encapsulation.

The sustained or controlled release of nitric oxide (NO) can be the most promising approach for the suppression or prevention of restenosis and thrombosis caused by stent implantation. The aim of this study is to investigate the feasibility in the potential use of layer-by-layer (LBL) coating with a NO donor-containing liposomes to control the release rate of NO from a metallic stent. Microscopic observation and surface characterizations of LBL-modified stents demonstrate successful LBL coating with liposomes on a stent. Release profiles of NO show that the release rate is sustained up to 5 d. In vitro cell study demonstrates that NO release significantly enhances endothelial cell proliferation, whereas it markedly inhibits smooth muscle cell proliferation. Finally, in vivo study conducted with a porcine coronary injury model proves the therapeutic efficacy of the NO-releasing stents coated by liposomal LBL technique, supported by improved results in luminal healing, inflammation, and neointimal thickening except thrombo-resistant effect. As a result, all these results demonstrate that highly optimized release rate and therapeutic dose of NO can be achieved by LBL coating and liposomal encapsulation, followed by significantly efficacious outcome in vivo.

Growth factors-loaded stents modified with hyaluronic acid and heparin for induction of rapid and tight re-endothelialization.

Rapid re-endothelialization of damaged vessel lining efficiently prevents restenosis and thrombosis and restores original vascular functions. In this study, we designed a novel metallic stent with a heparin-modified surface and used different methods, including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and divinyl sulfone (DVS), to load growth factors. First we loaded heparin into a dopamine-conjugated hyaluronic acid (HA) coating to serve as a growth factor reservoir. In a second step, we took advantage of the heparin-binding domain of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) to gain advanced re-endothelialization capabilities. We demonstrated that DVS technique offered higher amount of growth factor loading. In vitro assessment also showed better capillary-like structure formation and localized gap junctions when DVS coating was employed. This study suggested that growth factor loaded stent modified by HA and heparin provided the advantage to rapid and tight restoration of endothelium.

Artificial three-dimensional niches deconstruct pancreas development in vitro.

In the context of a cellular therapy for diabetes, methods for pancreatic progenitor expansion and subsequent differentiation into insulin-producing beta cells would be extremely valuable. Here we establish three-dimensional culture conditions in Matrigel that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the medium composition we generate either hollow spheres, which are mainly composed of pancreatic progenitors, or complex organoids that spontaneously undergo pancreatic morphogenesis and differentiation. The in vitro maintenance and expansion of pancreatic progenitors require active Notch and FGF signaling, thus recapitulating in vivo niche signaling interactions. Our experiments reveal new aspects of pancreas development, such as a community effect by which small groups of cells better maintain progenitor properties and expand more efficiently than isolated cells, as well as the requirement for three-dimensionality. Finally, growth conditions in chemically defined biomaterials pave the way for testing the biophysical and biochemical properties of the niche that sustains pancreatic progenitors.

Live mammalian cell arrays.

High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We developed a simple yet powerful method that uses contact spotting to generate high-density nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells.

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

High-throughput clonal analysis of neural stem cells in microarrayed artificial niches.

To better understand the extrinsic signals that control neural stem cell (NSC) fate, here we applied a microwell array platform which allows high-throughput clonal analyses of NSCs, cultured either as neurospheres or as adherent clones, exposed to poly(ethylene glycol) (PEG) hydrogel substrates functionalized with selected signaling molecules. We analyzed by time-lapse microscopy and retrospective immunostaining the role of integrin and Notch ligands, two key NSC niche components, in altering the behavior of several hundred single stem cells isolated from a previously described Hes5::GFP reporter mouse. NSC self-renewal was increased by 1.5-fold upon exposure to covalently tethered Laminin-1 and fibronectin fragment 9-10 (FN(9-10)), where 60-65% of single cells proliferated extensively and remained Nestin positive. Tethering of the Notch ligand Jagged-1 induced activation of Notch signaling. While Jagged-1 alone increased cell survival and proliferation, no further increase in the clonogenic potential of Hes5::GFP cells was observed upon co-stimulation with Laminin-1 and Jagged-1. We believe that the bioengineering of such in vitro niche analogues is a powerful approach to elucidate single stem cell fate regulation in a well-controlled fashion.