We developed a novel strategy to visualize and selectively deplete cell types in human organoid models, based on the favored DTR-mediated cell type depletion method that thus far was limited to mouse models. The complex multi-purpose gene knock-ins that we apply to human colon models, is applicable to virtually all cell types with well-defined marker genes. It is a powerful platform to investigate the cellular dynamics underlying regeneration in human tissues and to elucidate its molecular mechanisms with high temporal resolution.
What is the name of the methodology?
Cell-type ablation in human organoid models
OI Name
Hugo Snippert group
What is the methodology good for?
In stark contrast to genetic mouse models, the regenerative response upon stem cell damage is largely unexplored in human tissues. We made diphtheria toxin-mediated cell type ablation compatible with human cells, thereby enabling in-depth interrogation of the sequence of events during depletion and reappearance of stem cells in human organoid models of colon tissue.
What is/are the main advantages of this methodology over related technologies?
Despite the increased application of CRISPR-mediated genome editing, the number of successful attempts to visualize and manipulate stem cells by complex genetic knock-ins in normal human organoids remain limited.
We developed and successfully tested human LGR5 knock-in models akin to complex genetic mouse models, enabling combined a) visualization and b) depletion of human colon stem cells.
Regarding cell type depletion: unlike inducible Caspase9 which requires high expression levels to trigger apoptosis, we successfully modified the popular strategy in genetic mouse models that relies on the ectopic expression of Diphtheria Toxin Receptor (DTR). However, DTR is in fact the ubiquitously expressed human membrane-anchored glycoprotein HB-EGF that acts as the natural entry factor for Diphtheria Toxin (DT). To solve this challenge, we desensitize human organoids to DT by mutating HB-EGF in its DT binding domain by one amino acid substitution (E141H), phenocopying the non-sensitive mouse variant. The ‘murinized’ human organoids are compatible with DTR-mediated cell type ablation strategies using DTR-expression knock-in alleles.
What are the most important limitations of the methodology?
- Specific cell type marker genes are required to generate exclusive DTR expression
- We haven’t tested the system yet in multilayered organoids like brain (penetration DT across cell layers)
- Functional system requires two sequential CRISPR edits, which may not (yet) be feasible in primary cells with limited lifespan in vitro
What type of samples are compatible with methodology
Cancer cell lines | Primary cells in culture | Organoids | Primary tissue |
Yes | possibly | Yes | possibly |
What future develops to the methodology are you planning, in any?
We are developing ‘off-the-shelf’ protocols/vectors to generate the knock-ins in a wide-variety of cell types
If someone outside your lab wants to use the methodology, what is the best option?
A) Study our paper where we describe the generation of large complex knock-in alleles using Cas9 nickases, in particular the protocols in suppl. materials. (Bollen et al., PLOS Biol 2022).
B) Contact us for DNA plasmids/ constructs that can easily be modified for your gene-of-interest.
There is minimal ‘specific’ expertise/equipment required, as long there is expertise and equipment available for organoid cultures.
Name one or more people in your lab that are experienced with the methodology|
Defne Yalcin, PhD student in my lab.
Who originally developed the methodology?
Initiated by PhD student Joris Hageman. Initial manuscript is on BioRxiv (https://www.biorxiv.org/content/10.1101/2025.07.13.664589v1)