Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation

Understanding the different regulatory functions of epithelial and mesenchymal cell types in salivary gland development and cellular organization is essential for proper organoid formation and salivary gland tissue regeneration. Here, we demonstrate a biocompatible platform using pre-formed alginate hydrogel microtubes to facilitate direct epithelial–mesenchymal cell interaction for 3D salivary gland cell organization, which allows for monitoring cellular organization while providing a protective barrier from cell-cluster loss during medium changes. Using mouse salivary gland ductal epithelial SIMS cells as the epithelial model cell type and NIH 3T3 fibroblasts or primary E16 salivary mesenchyme cells as the stromal model cell types, self-organization from epithelial–mesenchymal interaction was examined. We observed that epithelial and mesenchymal cells undergo aggregation on day 1, cavitation by day 4, and generation of an EpCAM-expressing epithelial cell layer as early as day 7 of the co-culture in hydrogel microtubes, demonstrating the utility of hydrogel microtubes to facilitate heterotypic cell–cell interactions to form cavitated organoids. Thus, pre-formed alginate microtubes are a promising co-culture method for further understanding epithelial and mesenchymal interaction during tissue morphogenesis and for future practical applications in regenerative medicine.

Putting the Cell in Order

Organization is one of the most conspicuous features of cells. Not only are cells highly ordered (in the sense of regularity and predictability), but also they are organized: their order has purpose, or function. How does biological organization arise, and how is it transmitted from one generation to the next? A key element is genetic information encoded in DNA. Many scientists hold that DNA is the master molecule of life that prescribes all that cells are and do, and the general public has swallowed that doctrine whole. There is truth in this view of biological organization, inasmuch as genes do specify the chemical structure (and thereby the function) of proteins, nucleic acids, and (indirectly) many other biomolecules. But that is only part of an increasingly complex story. The higher levels of cell organization are not spelled out in the genes; they arise by self-organization, and are commonly transmitted to the next generation because the mother cell is architecturally continuous with its daughter. DNA provides an indispensable database, but does not direct the show. Organisms are better understood as complex interactive systems composed of genetically specified elements.

Functional and structural phenotyping of cardiomyocytes in the 3D organization of embryoid bodies exposed to arsenic trioxide

Chronic exposure to environmental pollutants threatens human health. Arsenic, a world-wide diffused toxicant, is associated to cardiac pathology in the adult and to congenital heart defects in the foetus. Poorly known are its effects on perinatal cardiomyocytes. Here, bioinformatic image-analysis tools were coupled with cellular and molecular analyses to obtain functional and structural quantitative metrics of the impairment induced by 0.1, 0.5 or 1.0 µM arsenic trioxide exposure on the perinatal-like cardiomyocyte component of mouse embryoid bodies, within their 3D complex cell organization. With this approach, we quantified alterations to the (a) beating activity; (b) sarcomere organization (texture, edge, repetitiveness, height and width of the Z bands); (c) cardiomyocyte size and shape; (d) volume occupied by cardiomyocytes within the EBs. Sarcomere organization and cell morphology impairment are paralleled by differential expression of sarcomeric α-actin and Tropomyosin proteins and of acta2, myh6 and myh7 genes. Also, significant increase of Cx40, Cx43 and Cx45 connexin genes and of Cx43 protein expression profiles is paralleled by large Cx43 immunofluorescence signals. These results provide new insights into the role of arsenic in impairing cytoskeletal components of perinatal-like cardiomyocytes which, in turn, affect cell size, shape and beating capacity.

High Resolution Single Cell Maps Reveals Distinct Cell Organization and Function Across Different Regions of the Human Intestine

The colon is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota, and affects overall health. To better understand its organization, functions, and its regulation at a single cell level, we performed CODEX multiplexed imaging, as well as single nuclear RNA and open chromatin assays across eight different intestinal sites of four donors. Through systematic analyses we find cell compositions differ dramatically across regions of the intestine, demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighborhoods and communities highlighting distinct immunological niches present in the intestine. We also map gene regulatory differences in these cells suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation, and organization for this organ, and serve as an important reference map for understanding human biology and disease.

Tau Regulates Glioblastoma Progression, 3D Cell Organization, Growth and Migration via the PI3K-AKT Axis

The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.

Ethical Dimensions of Human Organoids Research

Over the past decade, the development of three-dimensional mammalian cell organization—called human organoids—from stem cells has provided a framework for future clinical therapies. As human organoid research progresses, we also need to keep in mind the cross-cultural and ethical dimensions of human organoids research. Our review article aims to examine the ethical dimensions of cerebral human organoids and provide an ethical framework guide within human organoids research.

Mechanotransduction as a major driver of cell behaviour: mechanisms, and relevance to cell organization and future research

How do cells process environmental cues to make decisions? This simple question is still generating much experimental and theoretical work, at the border of physics, chemistry and biology, with strong implications in medicine. The purpose of mechanobiology is to understand how biochemical and physical cues are turned into signals through mechanotransduction. Here, we review recent evidence showing that (i) mechanotransduction plays a major role in triggering signalling cascades following cell–neighbourhood interaction; (ii) the cell capacity to continually generate forces, and biomolecule properties to undergo conformational changes in response to piconewton forces, provide a molecular basis for understanding mechanotransduction; and (iii) mechanotransduction shapes the guidance cues retrieved by living cells and the information flow they generate. This includes the temporal and spatial properties of intracellular signalling cascades. In conclusion, it is suggested that the described concepts may provide guidelines to define experimentally accessible parameters to describe cell structure and dynamics, as a prerequisite to take advantage of recent progress in high-throughput data gathering, computer simulation and artificial intelligence, in order to build a workable, hopefully predictive, account of cell signalling networks.

Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche

Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. We investigated the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We uncovered that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize an FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF-signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. The wing-disc produces two FGFs in distinct zones and restricts their signaling only through cytonemes. Consequently, although both FGFs use the same receptor, their cytoneme-mediated signaling asymmetrically distributes different muscle-specific AMPs into different FGF-producing niches. Loss of cytoneme-mediated adhesion and FGF-signaling promotes AMPs to lose niche occupancy, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.

Topological properties accurately predict cell division events and organization of Arabidopsis thaliana's shoot apical meristem

Cell division and the resulting changes to the cell organization affect the shape and functionality of all tissues. Thus, understanding the determinants of the tissue-wide changes imposed by cell division is a key question in developmental biology. Here, we use a network representation of live cell imaging data from shoot apical meristems (SAMs) in Arabidopsis thaliana to predict cell division events and their consequences at a tissue level. We show that a classifier based on the SAM network properties is predictive of cell division events, with validation accuracy of 82%, on par with that based on cell size alone. Further, we demonstrate that the combination of topological and biological properties, including: cell size, perimeter, distance, and shared cell wall between cells, can further boost the prediction accuracy of resulting changes in topology triggered by cell division. Using our classifiers, we demonstrate the importance of microtubule mediated cell-to-cell growth coordination in influencing tissue-level topology. Altogether, the results from our network-based analysis demonstrates a feedback mechanism between tissue topology and cell division in A. thaliana's SAMs.

3D Cell neighbour dynamics in growing pseudostratified epithelia

During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organize their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organization in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, i.e., Euler's formula, Lewis' law, and Aboav-Weaire's law, at all times and across the entire tissue thickness. This arrangement minimizes the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organization in growing epithelia emerges from simple physical principles.