NuclearIDTracker resolves intestinal cell identity and lineage dynamics through nuclear phenotypic signatures

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract Organoid models have transformed our understanding of intestinal renewal. Fluorescent imaging has been extensively used to identify key cell types and their differentiation pathways, but immunofluorescence provides only static readouts, whereas live imaging requires fluorescent-reporter engineering and is constrained by limited multiplexing and spectral overlap. Here, we introduce NuclearIDTracker, an explainable machine-learning framework that infers cell identity directly from 3D nuclear segmentations. Using a single nuclear marker, NuclearIDTracker accurately classifies intestinal cell types and integrates with single-cell tracking to resolve lineages and reconstruct dynamic state transitions during organoid development. We show that TA-like cells, rather than stem cells, drive early crypt formation and generate enterocyte and Paneth lineages, as well as the stem-cell population, which emerges only later and subsequently replenishes the TA-like compartment. Following stem-cell ablation, crypt regeneration was not driven by a single discrete cell type. Instead, multiple epithelial populations converged on a proliferative regenerative state with a nuclear phenotypic signature that resembled, but remained distinct from, that of homeostatic TA-like cells, and a YAP/TAZ-associated fetal-like transcriptional signature. Thus, nuclear phenotypic signatures resolve cell identity and reveal coordinated epithelial plasticity during crypt regeneration. NuclearIDTracker establishes a non-perturbative tool to quantify cell identity and state dynamics at single-cell resolution, revealing previously inaccessible biological dynamics and expanding the toolkit for studying epithelial homeostasis, regeneration, and disease.

Authors 12

1. Nguyen T.B. Nguyen

   University Medical Center Utrecht · Oncode Institute

   Affiliation as printed

   Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Heidelberglaan 100, 3584 CG Utrecht, the Netherlands

   Oncode Institute, The Netherlands

2. Rutger N.U. Kok

   University Medical Center Utrecht · Oncode Institute

   Affiliation as printed

   Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Heidelberglaan 100, 3584 CG Utrecht, the Netherlands

   Oncode Institute, The Netherlands

3. Sira Gevers

   University Medical Center Utrecht · Oncode Institute

   Affiliation as printed

   Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Heidelberglaan 100, 3584 CG Utrecht, the Netherlands

   Oncode Institute, The Netherlands

4. Xuan Zheng

   Royal Netherlands Academy of Arts and Sciences · University Medical Center Utrecht · Oncode Institute

   Affiliation as printed

   Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, University Medical Center Utrecht, the Netherlands

   Oncode Institute, The Netherlands

5. Max A. Betjes

   Institute for Atomic and Molecular Physics

   Affiliation as printed

   Autonomous Matter Department, AMOLF institute, Amsterdam, the Netherlands

6. Lukas Ritter

   University of Basel

   Affiliation as printed

   Medicine, University of Basel

7. Danny Feijtel Aachen Cell and Chemical Biology

   Leiden University Medical Center

   Affiliation as printed

   Cell and Chemical Biology, Leiden University Medical Center

8. Matthew B. Smith

   University Medical Center Utrecht

   Affiliation as printed

   Lab of Cellular Disease Models, Department of Paediatrics, Regenerative Medicine Centre Utrecht, UMC Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

9. Sam F.B. van Beuningen

   University Medical Center Utrecht

   Affiliation as printed

   Lab of Cellular Disease Models, Department of Paediatrics, Regenerative Medicine Centre Utrecht, UMC Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

10. Jeroen Sebastiaan van Zon Aachen Institute of Biology Leiden

    Leiden University · Institute for Atomic and Molecular Physics

    Affiliation as printed

    Autonomous Matter Department, AMOLF institute, Amsterdam, the Netherlands

    Institute of Biology Leiden, Leiden University, Leiden, The Netherlands

11. Sander J. Tans

    Institute for Atomic and Molecular Physics · Delft University of Technology

    Affiliation as printed

    Autonomous Matter Department, AMOLF institute, Amsterdam, the Netherlands

    Bionanoscience Department and Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, Netherlands

12. Maria J. Rodríguez Colman corresponding

    University Medical Center Utrecht · Oncode Institute

    Affiliation as printed

    Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Heidelberglaan 100, 3584 CG Utrecht, the Netherlands

    Oncode Institute, The Netherlands