Imaging Organoids, Exploiting New Dimensions in Cell Culture
The application of 3D-cell or “organoid” culture can reveal novel information about complex, tempro-spatially regulated developmental events such as patterning, signalling and disease progression. This session will highlight different ways that investigators have used imaging and data-analysis tools to gain unique insights into fundamental cell biology questions beyond what has been possible through traditional methods. Join us for what promises to be a fascinating and cutting edge session.
Patterning Principles of the Small Intestinal Crypt – Kara McKinley:
The mammalian small intestine exhibits a remarkable capacity for renewal during homeostasis and regeneration after injury. This capacity is powered by stem cells, which give rise to cells committed to executing the absorptive or secretory functions of the epithelium. I will discuss two studies using live imaging of organoids to understand the mechanisms that organize stem cells, secretory cells, and absorptive cells into the precise patterns required for tissue function. These studies reveal how cells integrate spatial and mechanical cues with fate decisions to precisely replace lost material during regeneration and renewal.
4D Cell Biology: Adaptive optics lattice light-sheet imaging and AI powered big data processing of live stem cell-derived organoids – Johannes Schoeneberg
New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome- edited human embryonic stem cells, which were differentiated into intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 μm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. We then expressed fluorescent protein fusions of actin and tubulin in genome-edited induced human pluripotent stem cells, which were differentiated into human cortical organoids. Using the AO-LLSM mode on the new MOSAIC (Multimodal Optical Scope with Adaptive Imaging Correction) allowed us to image neuronal migration deep in the organoid. We augmented pyLattice with a deep learning module and used it to process the brain organoid data.
Using Organoids to Understand Mechanisms and Consequences of Aging in Breast – Mark LaBarge:
Aging is overwhelmingly the main risk factor for carcinomas in humans, indeed, nearly 80% of all breast cancers are diagnosed in women over 50 years of age. Invertebrates lack mammary glands, and the relationship between age and mammary tumor incidence in mice differs significantly from humans. Examination of breast tissue in situ and in primary culture has revealed the luminal epithelia as a site of significant phenotypic changes with age that merits additional scrutiny because that is the region where breast cancer cells of origin reside. We utilize human mammary epithelial organoids that are reconstituted from a diverse bank of primary human mammary epithelial cells (HMEC) to examine the mechanisms and consequences of aging that lead to increased breast cancer susceptibility. Our 3D culture system enables highly parallel phenotypic analysis of thousands of reconstituted organoids for long periods of time, up to 200 days, in Matrigel-free conditions. Maintaining the luminal lineage in standard culture has been a persistent challenge for more than three decades, however, reconstituted multilineage organoids and bilayer cultures provided the microenvironment cues to maintain luminal cells for long-term. Heterochronus luminal‐myoepithelial organoids revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. Age‐driven changes in luminal‐myoepithelial cell gap‐ and tight‐junctions are implicated as key facilitators of the age‐related states. We provide evidence that the luminal lineage is exquisitely sensitive to microenvironment conditions, and that states of aging are cell-non‐autonomously communicated through microenvironment cues over at least one cell diameter.
