Single cell study for the population and signaling pathways of gastrointestinal stem cells niches

In the human body, the development of distinct organs is regulated by specific stromal niche (mesenchymal stem cells) signals. For the stomach and intestines, epithelial developments are controlled by the activities of stem cells from discrete niches: isthmus and crypt, respectively. To both organs, Wnt signaling is the signal transduction pathway that promotes tumorigenesis, regulates stem cell homeostasis and differentiation. Extensive studies have already digested the source of Wnt ligands from intestinal stromal cells (Paneth cells). Also, its transcriptional control through β-catenin was analyzed recently by our Nadia instrument. In contrast, little is known about the source of stomach Wnt ligands as well as their production.

A pre- print published by Kim et al., uses single cell transcriptomes analysis to identify a stem cell niche that is conserved between the stomach and intestines, and the role of stomach stromal cells, in particular. From the single cells pattern, they characterized these conserved stromal cells with telocyte, pericyte markers and a high expression of Wnt ligands. Furthermore, the Wnt signaling pathway directly mediated by Hedgehog (Hh-) and GLI2- transcriptional activation was also revealed. Lastly, through genetic analysis on mouse models, these conserved stromal Wnt signals show crucial roles in gut regeneration and development rather than in maintaining the stem cell niche’s homeostasis.

Let’s take a closer look at the results:

1. Single cell analysis identified conserved gastrointestinal stromal populations.
2. Wnt secretion by pericyte-like cells during regeneration.
3. Hh-GLI2 activation of Wnt signaling in conserved stromal cells.
4. Redundant role of stromal Wnt secretion in gut development.

Conserved gastrointestinal stromal populations:

To study heterogeneity, gastrointestinal stromal cells from mice were isolated and the transcriptomic profiles of 4946 stomach and 3459 intestinal single cells were analyzed by the Drop-Seq protocol with our Nadia instrument. t-SNE (T-distributed stochastic neighbor embedding) and unsupervised hierarchical data analysis distinguished 17 and 12 stromal cell clusters with either similar or distinct transcriptomic profiles between the stomach and intestines, respectively.

Then, the Markov affinity-based graph (MAGIC) was used to assess the Wnt ligand expression in the stem cell niche signaling. While the distinct stromal clusters also showed the Wnt ligand expression, a notable high enrichment of this signal was marked in the conserved niche of stomach and intestines. These conserved niches expressed pericyte markers (Ng2- Cspg4; Pdgfr-β) and telocyte markers (FoxL1) through fluorescence staining and hybridization. Therefore, they termed this conserved stromal niche as pericyte-like cells.


Wnt secretion by pericyte-like cells during regeneration

Accordingly, the function of these pericyte-like cells as a stem cell niche was assessed by their Wnt signal expression. Firstly, RNA fluorescence hybridization confirmed the expression of Wnt ligands (Wnt2b and Wnt4) in these cell populations. Then, to test its essence as a stem cell niche, they deleted the Wnt trans-membrane secretion protein Wntless (Wls). This resulted in a reduction of both stomach and intestinal stem cells, however, no proliferation changes in gastrointestinal was observed, suggesting alternative sources of Wnt signals. Therefore, these pericyte-like stromal cells were hypothesized to have a critical role in gastrointestinal regeneration. To evaluate this, they irradiated the gene-edited mice versus control mice and witnessed the malfunction in the regenerative progenitors and stem cells of both stomach and intestines at 10 days post-irradiation. Thus, we can clearly conclude that the pericyte-like stromal cells are critical for Wnt stromal niche regeneration.

Irradiation scheme: whole body irradiation (9Gy), Wls: Wntless


Hh-GLI2 activation of Wnt signaling in conserved stromal cells

The single cell RNA-seq data also demonstrated a high enrichment of Hh pathway in the conserved stromal cells. Only activated in the gut, Hh is an essential signal for the epithelial and stem cell proliferation which also regulates the Wnt ligands. To study its Wnt – regulating role, they activated the Hh signaling in the conserved pericyte-like stromal cells. As a result, the proliferation rate increased in both the gastrointestinal epithelial cells and their progenitors or stem cells. Meanwhile, fluorescence hybridization shows a higher level of Wnt ligands in these conserved stromal cells, strongly suggesting Hh activation of Wnt signaling. Interestingly, Hh downstream transcription factor – GLI2 was abundant in these conserved cells and highlighted for the enrichment of Hh and Wnt pathways. Thus, transcriptional reporter assay in cultured stomach and intestinal mesenchymal cells was performed to confirm the direct activation of GLI2 on stromal Wnt ligands (Wnt2b, Wnt9a).


Redundant role of stromal Wnt secretion in gut development

Due to the mild effect of Wnt inhibition on the stem cell and the ubiquity of Wnt ligands in other stromal cell populations, they propose a non – essential role of gastrointestinal stromal cells as a Wnt stem cell niche. To further clarify the functions of these gut stromal Wnt signals in development, they investigated the gastrointestinal phenotype of Wnt-deleted (Wlsfl/fl) mice. Significant reduction of the forestomach and intestinal lumen size coupled with epithelial depletion was shown through hematoxylin-eosin staining. Further study also demonstrated defects in stem cells development and epithelial proliferation as well as Wnt target gene expression. To conclude, they indicated a critical role for the gastrointestinal stromal Wnt niche during development but not for the stem cell niche homeostasis.

Example of stomach defects

Source: Kim et al. (2020)



In conclusion, this study disclosed the transcriptional profile and signaling pathway of a conserved gastrointestinal stromal stem cell niche as well as highlighted their redundancy among the stromal cell population. These pericyte-like cells regulate the regeneration and development of stomach and intestinal epithelial cells by expressing the Wnt signals activated by the Hh-GLI2 transcriptional factors. Single cell analysis by Drop-seq using the Nadia instrument have proven their efficacy in gastrointestinal niche cells clustering and enrichment with powerful data analysis such as the Seurat package.

If you are interested in studying stem cells and human health in general by single cell research, have a look at our solutions:

Single cell RNA-Seq with the Nadia platform.

RNAdia kit for Single Cell RNA-Seq on Nadia

Dolomite Bio also offers single-cell analysis service using in-house bioinformatics pipeline

Further reading