Significance: The pituitary is our master endocrine gland. Local damage and aging present important threats. We started to decrypt the ill-defined regulation of the gland’s stem cells, typically dormant but acutely activated upon damage. Single-cell transcriptomics uncovered interleukin-6 as a pituitary stem cell activator upon local damage, corroborated in vivo and in vitro using stem cell–derived organoids. This competence extinguishes at aging, concurrent with a raised inflammatory state in the older gland (inflammaging). However, the aging pituitary’s stem cells retain intrinsic activatability, resurfacing once released from their impeding tissue milieu. Our insights may instigate tactics to refrain the pituitary from aging or rejuvenate the aging gland. The single-cell transcriptomic database provides a powerful resource to decipher pituitary damage and aging.

Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2⁺ cells generating gastric spheroids differed from adult glandular Lgr5⁺ stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5⁺ stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2⁺ cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2⁺ cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/β-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/β-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.

The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b‐catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5‐eGFP‐IRES‐Cre and Lgr5‐DTReGFP embryos reveals that Lgr5 deficiency during Wnt‐mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses reveal that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5 antagonizes the Rspondin 2‐Wnt‐mediated response in ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISCs.

The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as short-chain fatty acids (SCFA). Here, we investigate the spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and study the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is predominantly detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we show that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by a reduced defense response to bacteria in colon crypts and slight dysbiosis. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Taken together, our work provides evidence that Olfr78 contributes to colon homeostasis by promoting enterochromaffin cell differentiation.

Cold atmospheric plasma (CAP) treatment has been proposed as a potentially innovative therapeutic tool in the biomedical field, notably for cancer due to its proposed toxic selectivity on cancer cells versus healthy cells. In the present study, we addressed the relevance of three-dimensional organoid technology to investigate the biological effects of CAP on normal epithelial stem cells and tumor cells isolated from mouse small intestine. CAP treatment exerted dose-dependent cytotoxicity on normal organoids and induced major transcriptomic changes associated with the global response to oxidative stress, fetal-like regeneration reprogramming, and apoptosis-mediated cell death. Moreover, we explored the potential selectivity of CAP on tumor-like Apc-deficient versus normal organoids in the same genetic background. Unexpectedly, tumor organoids exhibited higher resistance to CAP treatment, correlating with higher antioxidant activity at baseline as compared to normal organoids. This pilot study suggests that the ex vivo culture system could be a relevant alternative model to further investigate translational medical applications of CAP technology.