Identification of a Novel Lgr5-Independent Intestinal Lineage Involved in Epithelial Regeneration

Core Concepts

A distinct lineage of quiescent intestinal stem cells, independent of the Lgr5-positive crypt base columnar cells, contributes to epithelial regeneration following injury.

Abstract

The study demonstrates the existence of a previously uncharacterized intestinal cell lineage that is distinct from the Lgr5-positive crypt base columnar cells (CBCs), the well-established intestinal stem cells. This novel lineage can be identified by its lack of recombination in VillinCre transgenic mice.

Key findings:

1. Collagenase/dispase treatment of adult mouse intestine releases cells that can be cultured as immortal, cystic spheroids in matrigel. These spheroids display a fetal-like transcriptional profile, with downregulation of CBC markers like Lgr5, Ascl2, Smoc2, and Olfm4.
2. Lineage tracing experiments show that the spheroid-generating cells belong to a developmental lineage independent of Lgr5-positive CBCs.
3. Following ablation of CBCs using a diphtheria toxin model, VillinCre-negative regenerating crypts emerge, demonstrating the involvement of this novel lineage in epithelial regeneration.
4. scRNA-seq analysis identifies the spheroid-generating cells as a quiescent, Olfm4-positive population distinct from the actively cycling CBCs.

These results suggest that in addition to the known plasticity of differentiated intestinal cell types, a hierarchical stem cell model involving this Lgr5-independent lineage also contributes to intestinal regeneration.

Stats

The number of caspase 3-positive cells in crypt cells peaked at 24 hours after tamoxifen administration in VilCreERT2/Mcl1fl/fl mice.
The yield of organoids from the EDTA fraction was reduced to zero 24 hours after tamoxifen treatment in VilCreERT2/Mcl1fl/fl mice, indicating effective ablation of CBCs.
The proportion of VillinCre-negative crypts increased from around 30% in untreated VilCre/Lgr5-DTR/Rosa26Tomato mice to over 60% in mice treated with diphtheria toxin.

Quotes

"Lineage tracing experiments demonstrate that the cells at the origin of these spheroids belong to a separate developmental lineage, independent of Lgr5+ve CBCs, and are involved in regeneration of the epithelium following ablation of CBCs."
"Together, these results suggest the existence of peculiar stem cells, tightly attached to extracellular matrix, which activate a regeneration program when they are severed from their normal environment by collagenase/dispase treatment and cultured in matrigel."
"Our results lead to the conclusion that a hierarchical stem cell model applies to regeneration of the intestinal epithelium, in addition to the plasticity model."

Key Insights Distilled From

An Lgr5-independent developmental lineage is involved in mouse intestinal regeneration

by Marefati,M.,... at www.biorxiv.org 03-13-2024

https://www.biorxiv.org/content/10.1101/2024.03.11.584399v2

Deeper Inquiries

What are the specific molecular markers that can identify the Lgr5-independent intestinal stem cell lineage in the homeostatic epithelium?

In the homeostatic epithelium, the Lgr5-independent intestinal stem cell lineage can be identified by specific molecular markers such as Olfm4. Olfm4 is a marker that is commonly associated with quiescent stem cells in the intestine. Additionally, the Lgr5-independent lineage may exhibit an abundance of ribosomal protein transcripts, which is a characteristic feature of stem cells. These cells may also show downregulation of proliferative markers, indicating their quiescent state. The presence of nuclear-encoded mitochondrial proteins, including complex IV cytochrome oxidases, mitochondrial ATP synthase subunits, and the ATPase inhibiting factor, may also serve as markers for this unique stem cell lineage.

How does the quiescent state of the Lgr5-independent stem cells contribute to their role in regeneration compared to the actively cycling Lgr5-positive CBCs?

The quiescent state of the Lgr5-independent stem cells plays a crucial role in regeneration compared to the actively cycling Lgr5-positive CBCs. While Lgr5-positive CBCs are known for their active proliferation and role in daily epithelium maintenance, the quiescent state of the Lgr5-independent stem cells allows them to remain dormant until needed for regeneration. This quiescent state enables them to conserve energy and resources, making them well-suited for long-term tissue repair and regeneration processes. Additionally, the quiescent nature of these stem cells may protect them from DNA damage and mutations that can occur during active cell division, ensuring the integrity of the stem cell pool for future regeneration events.

Could the Lgr5-independent lineage also play a role in intestinal homeostasis beyond regeneration, and how might this be regulated?

The Lgr5-independent lineage may indeed play a role in intestinal homeostasis beyond regeneration. These quiescent stem cells could serve as a reserve pool of stem cells that are ready to be activated in response to various stresses or injuries that disrupt the normal intestinal epithelium. In homeostasis, these cells may remain dormant but poised to proliferate and differentiate when needed to maintain tissue integrity.
The regulation of the Lgr5-independent lineage in intestinal homeostasis could be influenced by various factors. Environmental cues, signaling molecules, and interactions with the surrounding microenvironment may play a role in activating or maintaining the quiescent state of these stem cells. Additionally, intrinsic factors such as epigenetic modifications and transcriptional regulation may control the transition between quiescence and activation in response to tissue demands. Further research is needed to elucidate the precise mechanisms that regulate the Lgr5-independent lineage in intestinal homeostasis.