4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell–derived intestinal organoids

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 hESC-derived 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. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.

Download Full-text

4D Cell Biology: Big Data Image Analytics and Lattice Light-Sheet Imaging Reveal Dynamics of Clathrin-Mediated Endocytosis in Stem Cell-Derived Intestinal Organoids

Biophysical Journal ◽

10.1016/j.bpj.2018.11.928 ◽

2019 ◽

Vol 116 (3) ◽

pp. 167a

Author(s):

Johannes Schöneberg ◽

Daphné Dambournet ◽

Tsung-Li Liu ◽

Ryan Forster ◽

Dirk Hockemeyer ◽

...

Keyword(s):

Big Data ◽

Stem Cell ◽

Cell Biology ◽

Light Sheet ◽

Intestinal Organoids

Download Full-text

4D cell biology: Big data image analytics and lattice light‐sheet imaging reveal dynamics of clathrin‐mediated endocytosis in stem cell‐derived intestinal organoids

The FASEB Journal ◽

10.1096/fasebj.2019.33.1_supplement.659.3 ◽

2019 ◽

Vol 33 (S1) ◽

Author(s):

Johannes Schöneberg ◽

Daphné Dambournet ◽

Tsung‐Li Liu ◽

Ryan Forster ◽

Dirk Hockemeyer ◽

...

Keyword(s):

Big Data ◽

Stem Cell ◽

Cell Biology ◽

Light Sheet ◽

Intestinal Organoids

Download Full-text

Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

AJP Cell Physiology ◽

10.1152/ajpcell.00188.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. C725-C736

Author(s):

Gurbind Singh ◽

Divya Sridharan ◽

Mahmood Khan ◽

Polani B. Seshagiri

Keyword(s):

Cell Biology ◽

Fluorescent Protein ◽

Es Cells ◽

Monochromatic Light ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Medical Diagnostics ◽

Calcium Transients ◽

Egfp Expression ◽

Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

Download Full-text

Lineage specification of early embryos and embryonic stem cells at the dawn of enabling technologies

National Science Review ◽

10.1093/nsr/nwx093 ◽

2017 ◽

Vol 4 (4) ◽

pp. 533-542 ◽

Cited By ~ 3

Author(s):

Guangdun Peng ◽

Patrick P. L. Tam ◽

Naihe Jing

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Biology ◽

Developmental Process ◽

Embryonic Stem ◽

Molecular Signaling ◽

Lineage Specification ◽

Genomic Technologies ◽

Enabling Technologies ◽

Stem Cell Pluripotency

Abstract Establishment of progenitor cell populations and lineage diversity during embryogenesis and the differentiation of pluripotent stem cells is a fascinating and intricate biological process. Conceptually, an understanding of this developmental process provides a framework to integrate stem-cell pluripotency, cell competence and differentiating potential with the activity of extrinsic and intrinsic molecular determinants. The recent advent of enabling technologies of high-resolution transcriptome analysis at the cellular, population and spatial levels proffers the capability of gaining deeper insights into the attributes of the gene regulatory network and molecular signaling in lineage specification and differentiation. In this review, we provide a snapshot of the emerging enabling genomic technologies that contribute to the study of development and stem-cell biology.

Download Full-text

First person – Federico Pecori

Journal of Cell Science ◽

10.1242/jcs.255166 ◽

2020 ◽

Vol 133 (20) ◽

pp. jcs255166

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Cell Biology ◽

Embryonic Stem ◽

Early Career ◽

First Person ◽

Receptor Endocytosis ◽

Early Career Researchers ◽

Selection Of

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Federico Pecori is first author on ‘Mucin-type O-glycosylation controls pluripotency in mouse embryonic stem cells via Wnt receptor endocytosis’, published in JCS. Federico is a PhD student in the lab of Shoko Nishihara at the Laboratory of Cell Biology, Department of Bioinformatics, Soka University, Tokyo, Japan, where he is interested in the mechanisms regulating stem cell identity.

Download Full-text

IMAGING REDOX STATE HETEROGENEITY WITHIN INDIVIDUAL EMBRYONIC STEM CELL COLONIES

Journal of Innovative Optical Health Sciences ◽

10.1142/s1793545811001617 ◽

2011 ◽

Vol 04 (03) ◽

pp. 279-288 ◽

Cited By ~ 4

Author(s):

HE N. XU ◽

RUSSELL C. ADDIS ◽

DAVIDA F. GOINGS ◽

SHOKO NIOKA ◽

BRITTON CHANCE ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Redox State ◽

Embryonic Stem ◽

Reduced Form ◽

Significant Heterogeneity ◽

Imaging Data ◽

Mitochondrial Redox State ◽

Reduced State

Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/(Fp + NADH)), where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidized flavoproteins, has been established as a sensitive indicator of mitochondrial redox state. In this paper, we report our redox imaging data on the mitochondrial redox state of mouse ESC (mESC) colonies and the implications thereof. The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse embryonic fibroblasts (MEFs) on glass cover slips. The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies (size: ~200–440 μm), exhibiting a core with a more reduced state than the periphery. This more reduced state positively correlates with the expression pattern of Oct4, a well-established marker of pluripotency. Our observation is the first to show the heterogeneity in the mitochondrial redox state within a mESC colony, suggesting that mitochondrial redox state should be further investigated as a potential new biomarker for the stemness of embryonic stem cells.

Download Full-text

Myocardial aging and embryonic stem cell biology

Stem Cells: A Cellular Fountain of Youth - Advances in Cell Aging and Gerontology ◽

10.1016/s1566-3124(02)09047-8 ◽

2002 ◽

pp. 141-176 ◽

Cited By ~ 4

Author(s):

Kenneth R. Boheler ◽

Anna M. Wobus

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Biology ◽

Embryonic Stem ◽

Stem Cell Biology ◽

Myocardial Aging

Download Full-text

Interview with 2020 Hooke medal winner Ian Chambers

Journal of Cell Science ◽

10.1242/jcs.254219 ◽

2020 ◽

Vol 133 (20) ◽

pp. jcs254219

Keyword(s):

Stem Cell ◽

Stem Cell Research ◽

Cell Biology ◽

Embryonic Stem ◽

British Society ◽

Control Of Gene Expression ◽

Erythroid Precursor ◽

Cell Fate Decisions ◽

University Of Edinburgh ◽

The University

ABSTRACTIan Chambers studied biochemistry at the University of Strathclyde in Glasgow, UK. He then did his PhD in the laboratory of Paul Harrison at the Beatson Institute for Cancer Research, also in Glasgow. Ian studied the control of gene expression during the differentiation of erythroid precursor cells, discovering that the amino acid selenocysteine is encoded by UGA, which until then was thought to work only as a termination codon. Ian did his post-doctoral work on the regulation of the human immunodeficiency virus (HIV) with Paul Berg at Stanford University in California, USA. In 1991, he returned to Scotland to work on stem cell regulation with Austin Smith at the Centre for Genome Research (later the Institute for Stem Cell Research) at the University of Edinburgh, UK. During that time, Ian identified the transcription factor Nanog, which directs efficient embryonic stem cell self-renewal. Ian started his research group in 2006 at the University of Edinburgh, where he is also a Professor of Pluripotent Stem Cell Biology. His laboratory tries to understand the regulatory networks and transcription factors that control the identity of pluripotent embryonic stem cells, and how these modulate cell fate decisions during the differentiation process. Ian is now the Head of the Institute for Stem Cell Research at University of Edinburgh, an EMBO member and a Fellow of the Royal Society of Edinburgh. Ian is the recipient of the 2020 Hooke Medal from the British Society for Cell Biology (BSCB).

Download Full-text