HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal to noise fluorescence

The expanded application of fluorescence imaging in biomedical and biological research towards more complex systems and geometries requires tools that can analyze a multitude of components at widely varying time- and length-scales. The major challenge in such complex imaging experiments is to cleanly separate multiple fluorescent labels with overlapping spectra from one another and background autofluorescence, without perturbing the sample with high levels of light. Thus, there is a requirement for efficient and robust analysis tools capable of quantitatively separating these signals. In response, we have combined multispectral fluorescence microscopy with hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). Here we demonstrate its capabilities in the dynamic imaging of multiple fluorescent labels in live, developing zebrafish embryos. HyU is more sensitive to low light levels of fluorescence compared to conventional linear unmixing approaches, permitting better multiplexed volumetric imaging over time, with less bleaching. HyU can also simultaneously image both bright exogenous and dim endogenous labels because of its high dynamic range. This allows studies of cellular behaviors, tagged components, and cell metabolism within the same specimen, offering a powerful window into the orchestrated complexity of biological systems.

Toward Multiplexed Optogenetic Circuits

Owing to its ubiquity and easy availability in nature, light has been widely employed to control complex cellular behaviors. Light-sensitive proteins are the foundation to such diverse and multilevel adaptive regulations in a large range of organisms. Due to their remarkable properties and potential applications in engineered systems, exploration and engineering of natural light-sensitive proteins have significantly contributed to expand optogenetic toolboxes with tailor-made performances in synthetic genetic circuits. Progressively, more complex systems have been designed in which multiple photoreceptors, each sensing its dedicated wavelength, are combined to simultaneously coordinate cellular responses in a single cell. In this review, we highlight recent works and challenges on multiplexed optogenetic circuits in natural and engineered systems for a dynamic regulation breakthrough in biotechnological applications.

Glial Patchwork: Oligodendrocyte Progenitor Cells and Astrocytes Blanket the Central Nervous System

Tiling is a developmental process where cell populations become evenly distributed throughout a tissue. In this review, we discuss the developmental cellular tiling behaviors of the two major glial populations in the central nervous system (CNS)—oligodendrocyte progenitor cells (OPCs) and astrocytes. First, we discuss OPC tiling in the spinal cord, which is comprised of the three cellular behaviors of migration, proliferation, and contact-mediated repulsion (CMR). These cellular behaviors occur simultaneously during OPC development and converge to produce the emergent behavior of tiling which results in OPCs being evenly dispersed and occupying non-overlapping domains throughout the CNS. We next discuss astrocyte tiling in the cortex and hippocampus, where astrocytes migrate, proliferate, then ultimately determine their exclusive domains by gradual removal of overlap rather than sustained CMR. This results in domains that slightly overlap, allowing for both exclusive control of “synaptic islands” and astrocyte-astrocyte communication. We finally discuss the similarities and differences in the tiling behaviors of these glial populations and what remains unknown regarding glial tiling and how perturbations to this process may impact injury and disease.

Electrical Stimulation and Cellular Behaviors in Electric Field in Biomedical Research

Research on the cellular response to electrical stimulation (ES) and its mechanisms focusing on potential clinic applications has been quietly intensified recently. However, the unconventional nature of this methodology has fertilized a great variety of techniques that make the interpretation and comparison of experimental outcomes complicated. This work reviews more than a hundred publications identified mostly from Medline, categorizes the techniques, and comments on their merits and weaknesses. Electrode-based ES, conductive substrate-mediated ES, and noninvasive stimulation are the three principal categories used in biomedical research and clinic. ES has been found to enhance cell proliferation, growth, migration, and stem cell differentiation, showing an important potential in manipulating cellular activities in both normal and pathological conditions. However, inappropriate parameters or setup can have negative effects. The complexity of the delivered electric signals depends on how they are generated and in what form. It is also difficult to equate one set of parameters with another. Mechanistic studies are rare and badly needed. Even so, ES in combination with advanced materials and nanotechnology is developing a strong footing in biomedical research and regenerative medicine.

Regulating the production and biological function of small extracellular vesicles: current strategies, applications and prospects

Numerous studies have confirmed the great application potentials of small extracellular vesicles (sEVs) in biological medical field, especially in tissue repair and regeneration. However, the production capability of sEVs by noncancerous cells is very limited, while their dosage requirements in disease treatments are usually very high. Meanwhile, as cell aging, the sEV production capability of cells decreases and the biological function of sEVs changes accordingly. In addition, for special applications, sEVs carrying desired bioactive substances should be designed to perform their expected biological function. Therefore, improving the production of sEVs and precisely regulating their biological function are of great significance for promoting the clinical applications of sEVs. In this review, some of the current classic strategies in affecting the cellular behaviors of donor cells and subsequently regulating the production and biological function of their sEVs are summarized, including gene engineering methods, stress-inducing conditions, chemical regulators, physical methods, and biomaterial stimulations. Through applying these strategies, increased yield of sEVs with required biological function can be obtained for disease treatment and tissue repair, such as bone regeneration, wound healing, nerve function recovery and cancer treatment, which could not only reduce the harvest cost of sEV but promote the practical applications of sEVs in clinic. Graphical Abstract

Mechanics of cell integration in vivo

During embryonic development, regeneration and homeostasis, cells have to physically integrate into their target tissues, where they ultimately execute their function. Despite a significant body of research on how mechanical forces instruct cellular behaviors within the plane of an epithelium, very little is known about the mechanical interplay at the interface between migrating cells and their surrounding tissue, which has its own dynamics, architecture and identity. Here, using quantitative in vivo imaging and molecular perturbations, together with a theoretical model, we reveal that multiciliated cell (MCC) precursors in the Xenopus embryo form dynamic filopodia that pull at the vertices of the overlying epithelial sheet to probe their stiffness and identify the preferred positions for their integration into the tissue. Moreover, we report a novel function for a structural component of vertices, the lipolysis-stimulated lipoprotein receptor (LSR), in filopodia dynamics and show its critical role in cell intercalation. Remarkably, we find that pulling forces equip the MCCs to remodel the epithelial junctions of the neighboring tissue, enabling them to generate a permissive environment for their integration. Our findings reveal the intricate physical crosstalk at the cell-tissue interface and uncover previously unknown functions for mechanical forces in orchestrating cell integration.

Roles of Integrins in Gastrointestinal Cancer Metastasis

Integrins are a large family of heterodimeric transmembrane receptors which mediate cell adhesion and transmit signals to the cell interior. The mechanistic roles of integrins have long been an enigma in cancer, given its complexity in regulating different cellular behaviors. Recently, however, increasing research is providing new insights into its function and the underlying mechanisms, which collectively include the influences of altered integrin expression on the aberrant signaling pathways and cancer progression. Many studies have also demonstrated the potentiality of integrins as therapeutic targets in cancer treatment. In this review, we have summarized these recent reports and put a particular emphasis on the dysregulated expression of integrins and how they regulate related signaling pathways to facilitate the metastatic progression of gastrointestinal cancer, including gastric cancer (GC) and colorectal cancer (CRC), which will address the crucial roles of integrins in gastrointestinal cancer.

Development and Utilization of Multifunctional Polymeric Scaffolds for the Regulation of Physical Cellular Microenvironments

Polymeric biomaterials exhibit excellent physicochemical characteristics as a scaffold for cell and tissue engineering applications. Chemical modification of the polymers has been the primary mode of functionalization to enhance biocompatibility and regulate cellular behaviors such as cell adhesion, proliferation, differentiation, and maturation. Due to the complexity of the in vivo cellular microenvironments, however, chemical functionalization alone is usually insufficient to develop functionally mature cells/tissues. Therefore, the multifunctional polymeric scaffolds that enable electrical, mechanical, and/or magnetic stimulation to the cells, have gained research interest in the past decade. Such multifunctional scaffolds are often combined with exogenous stimuli to further enhance the tissue and cell behaviors by dynamically controlling the microenvironments of the cells. Significantly improved cell proliferation and differentiation, as well as tissue functionalities, are frequently observed by applying extrinsic physical stimuli on functional polymeric scaffold systems. In this regard, the present paper discusses the current state-of-the-art functionalized polymeric scaffolds, with an emphasis on electrospun fibers, that modulate the physical cell niche to direct cellular behaviors and subsequent functional tissue development. We will also highlight the incorporation of the extrinsic stimuli to augment or activate the functionalized polymeric scaffold system to dynamically stimulate the cells.

Analysis of growth cone extension in standardized coordinates highlights self-organization rules during wiring of the Drosophila visual system

A fascinating question in neuroscience is how ensembles of neurons, originating from different locations, extend to the proper place and by the right time to create precise circuits. Here, we investigate this question in the Drosophila visual system, where photoreceptors re-sort in the lamina to form the crystalline-like neural superposition circuit. The repeated nature of this circuit allowed us to establish a data-driven, standardized coordinate system for quantitative comparison of sparsely perturbed growth cones within and across specimens. Using this common frame of reference, we investigated the extension of the R3 and R4 photoreceptors, which is the only pair of symmetrically arranged photoreceptors with asymmetric target choices. Specifically, we found that extension speeds of the R3 and R4 growth cones are inherent to their cell identities. The ability to parameterize local regularity in tissue organization facilitated the characterization of ensemble cellular behaviors and dissection of mechanisms governing neural circuit formation.