Biology subjects

Thirstrup, D. J.

Publications and source records attributed to Thirstrup, D. J..

4 recordsLinked to original sources

bioRxiv2024

A human induced pluripotent stem (hiPS) cell model for the holistic study of epithelial to mesenchymal transitions (EMTs)

The epithelial to mesenchymal transition (EMT) is a widely studied but poorly defined state change due to the variety of ways in which it has been characterized in cells. There is a need for reproducible cell model systems that enable the integration and comparison of different types of measured observations of cells across many distinct cellular contexts. We present human induced pluripotent stem (hiPS) cells as such a model system by demonstrating its utility through a comparative analysis of hiPS cell-EMT in 2D and 3D cell culture geometries. We developed live-imaging-based assays to directly compare examples of changes in cell function (via migration timing), molecular components (via expression of marker proteins), organization (via reorganization of cell junctions), and environment (via dynamics of basement membrane) in the same experimental system. The EMT-related changes we measured occurred earlier in 2D colonies than in 3D lumenoids, likely due to differences in the basement membrane environments associated with 2D vs. 3D initial hiPS cell culture geometries. We have made the 449 60-hour-long 3D time-lapse movies and the associated tools used for analysis and visualization open-source and easily accessible as a resource for future work in this field.

cell biology↗

bioRxiv2024

Colony context and size-dependent compensation mechanisms give rise to variations in nuclear growth trajectories

To investigate the fundamental question of how cellular variations arise across spatiotemporal scales in a population of identical healthy cells, we focused on nuclear growth in hiPS cell colonies as a model system. We generated a 3D timelapse dataset of thousands of nuclei over multiple days, and developed open-source tools for image and data analysis and an interactive timelapse viewer for exploring quantitative features of nuclear size and shape. We performed a data-driven analysis of nuclear growth variations across timescales. We found that individual nuclear volume growth trajectories arise from short timescale variations attributable to their spatiotemporal context within the colony. We identified a strikingly time-invariant volume compensation relationship between nuclear growth duration and starting volume across the population. Notably, we discovered that inheritance plays a crucial role in determining these two key nuclear growth features while other growth features are determined by their spatiotemporal context and are not inherited.

cell biology↗

bioRxiv2021

Mapping Protein Numbers in Living Cells

We introduce a new, robust method to map the numbers of proteins in living cells. The method can be applied to laser scanning, spinning disk, and lattice light-sheet microscopes in a robust, reproducible, and scalable fashion. The method uses calibrated EGFP solutions that are imaged with the appropriate microscope modality to create a calibration curve that is then applied to convert the fluorescence intensities from 3D microscope images into molecule numbers. We applied this method to human induced pluripotent stem cells in which proteins representing key cellular structures were endogenously tagged with mEGFP. We used the ratio of mEGFP-tagged proteins to total proteins to create 3D maps of live cells showing the density of total proteins measured in molecules per {micro}m3. The method opens the door to new quantitative single cell analyses of cellular protein numbers in the context of single cell gene expression, associations with cellular complexes, and changes in cellular behaviors. The method is capable of quantifying protein numbers, over three orders of magnitude, in the cytoplasm or within various cellular structures while offering the unique advantages of each microscopy modality.

cell biology↗

bioRxiv2020

Automated hiPSC culture and sample preparation for 3D live cell microscopy

Our goal is to identify and understand cellular behaviors using 3D live imaging of cell organization. To do this, we image human inducible pluripotent stem cell (hiPSC) lines expressing fluorescently tagged protein representing specific cellular organelles and structures. To produce large numbers of standardized cell images, we developed an automated hiPSC culture procedure, to maintain, passage and Matrigel coat 6-well plastic plates and 96-well glass plates compatible with high-resolution 3D microscopy. Here we describe this system including optimization procedures and specific values for plate movement, angle of tips, speed of aspiration and dispense, seeding strategies and timing of every step. We validated this approach through a side-by-side comparison of quality control results obtained from manual and automated methods. Additionally, we developed an automated image-based colony segmentation and feature extraction pipeline to predict cell count and select wells with consistent morphology for high resolution 3D microscopy.

cell biology↗