Biology subjects

Krueger, D.

Publications and source records attributed to Krueger, D..

5 recordsLinked to original sources

bioRxiv2024

Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets

Matrigel/BME, a basement membrane-like preparation, supports long-term growth of epithelial 3D organoids from adult stem cells (ASC)1,2. Here, we show that interaction between Matrigels major component Laminin111 with epithelial 6{beta}1-integrin is crucial for this process. The outer membrane protein Invasin of Yersinia is known to activate multiple integrin-{beta}1 complexes, including integrin-6{beta}1. A C-terminal integrin-binding fragment of Invasin, coated on culture plates, mediated gut epithelial cell adhesion. Addition of organoid growth factors allowed multi-passage expansion in 2D. Polarization, junction formation and generation of enterocytes, goblet cells, Paneth cells, and enteroendocrine cells was stable over time. Sustained expansion of other human-, mouse-, and even snake epithelia was accomplished under comparable conditions. The 2D organoid sheet format holds advantages over the 3D in gel format in terms of imaging, accessibility of basal and apical domains and automation for high throughput screening. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel/BME.

cell biology↗

bioRxiv2024

Tuft cells act as regenerative stem cells in the human intestine

In mice, intestinal tuft cells have been described as a long-lived, post-mitotic cell type of which two distinct subsets have been identified, named tuft-1 and tuft-21. By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase upon interleukin (IL)-4 and IL-13 exposure, as reported previously in mouse2-4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem- and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

cell biology↗

bioRxiv2021

Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

Human pluripotent stem cell derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for derivation of skeletal muscle cells and their utility in three-dimensional skeletal muscle organoid formation as well as skeletal muscle tissue engineering. Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors of hypaxial origin followed by primary and secondary fetal myogenesis into hypaxial muscle with development of a satellite cell pool and evidence for innervation in vitro. Skeletal muscle organoids faithfully recapitulate all steps of embryonic myogenesis in 3D Tissue engineered muscle exhibits organotypic maturation and function, advanced by thyroid hormone. Regenerative competence was demonstrated in a cardiotoxin injury model with evidence of satellite cell activation as underlying mechanism. Collectively, we introduce a hypaxial muscle model with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease, and repair.

cell biology↗

bioRxiv2021

Cell division in tissues enables macrophage infiltration

Migration of cells through diverse tissues is essential for development, immune response and cancer metastasis 1-3. To reach their destination, cells must overcome the resistance imposed by complex microenvironments, composed of neighboring cells and extracellular matrix (ECM)4-6. While migration through pores and tracks in ECM has been well studied 4,5,7, little is known about cellular traversal into confining cell-dense tissues. Here by combining quantitative live imaging with genetic and optogenetic perturbations we identify a crucial role for cell division during cell migration into tissues. We find that normal embryonic invasion by Drosophila macrophages between the ectoderm and mesoderm8,9 absolutely requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by Integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade. Decreasing or increasing the frequency of ectodermal division correspondingly either hinders or promotes macrophage invasion. Reducing the levels of focal adhesion components in the ectoderm allows macrophage entry even in the absence of division. Our study demonstrates the critical importance of division at the entry site to enable in vivo cell invasion by relieving the steric impediment caused by focal adhesions. We thus provide a new perspective on the regulation of cellular movement into tissues.

cell biology↗

bioRxiv2019

Optogenetic inhibition of Delta reveals digital Notch signaling output during tissue differentiation

Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue- and cell-scale regulatory modes. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.

developmental biology↗