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Abstract The human embryo is a complex structure that emerges and develops as a result of cell-level decisions guided by both intrinsic genetic programs and cell–cell interactions. Given limited accessibility and associated ethical constraints of human embryonic tissue samples, researchers have turned to the use of human stem cells to generate embryo models to study specific embryogenic developmental steps. However, to study complex self-organizing developmental events using embryo models, there is a need for computational and imaging tools for detailed characterization of cell-level dynamics at the single cell level. In this work, we obtained live cell imaging data from a human pluripotent stem cell (hPSC)-based epiblast model that can recapitulate the lumenal epiblast cyst formation soon after implantation of the human blastocyst. By processing imaging data with a Python pipeline that incorporates both cell tracking and event recognition with the use of a CNN-LSTM machine learning model, we obtained detailed temporal information of changes in cell state and neighborhood during the dynamic growth and morphogenesis of lumenal hPSC cysts. The use of this tool combined with reporter lines for cell types of interest will drive future mechanistic studies of hPSC fate specification in embryo models and will advance our understanding of how cell-level decisions lead to global organization and emergent phenomena. Insight, innovation, integration: Human pluripotent stem cells (hPSCs) have been successfully used to model and understand cellular events that take place during human embryogenesis. Understanding how cell–cell and cell–environment interactions guide cell actions within a hPSC-based embryo model is a key step in elucidating the mechanisms driving system-level embryonic patterning and growth. In this work, we present a robust video analysis pipeline that incorporates the use of machine learning methods to fully characterize the process of hPSC self-organization into lumenal cysts to mimic the lumenal epiblast cyst formation soon after implantation of the human blastocyst. This pipeline will be a useful tool for understanding cellular mechanisms underlying key embryogenic events in embryo models.
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An optimized pipeline for live imaging whole Arabidopsis leaves at cellular resolution
Abstract BackgroundLive imaging is the gold standard for determining how cells give rise to organs. However, tracking many cells across whole organs over large developmental time windows is extremely challenging. In this work, we provide a comparably simple method for confocal live imaging entireArabidopsis thalianafirst leaves across early development. Our imaging method works for both wild-type leaves and the complex curved leaves of thejaw-1Dmutant. ResultsWe find that dissecting the cotyledons, affixing a coverslip above the samples and mounting samples with perfluorodecalin yields optimal imaging series for robust cellular and organ level analysis. We provide details of our complementary image processing steps in MorphoGraphX software for segmenting, tracking lineages, and measuring a suite of cellular properties. We also provide MorphoGraphX image processing scripts we developed to automate analysis of segmented images and data presentation. ConclusionsOur imaging techniques and processing steps combine into a robust imaging pipeline. With this pipeline we are able to examine important nuances in the cellular growth and differentiation ofjaw-Dversus WT leaves that have not been demonstrated before. Our pipeline is approachable and easy to use for leaf development live imaging.
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- Award ID(s):
- 2203275
- PAR ID:
- 10394871
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Plant Methods
- Volume:
- 19
- Issue:
- 1
- ISSN:
- 1746-4811
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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