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Stem cells in the adult pituitary are quiescent yet show acute activation upon tissue injury. The molecular mechanisms underlying this reaction are completely unknown. We applied single-cell transcriptomics to start unraveling the acute pituitary stem cell activation process as occurring upon targeted endocrine cell–ablation damage. This stem cell reaction was contrasted with the aging (middle-aged) pituitary, known to have lost damage-repair capacity. Stem cells in the aging pituitary show regressed proliferative activation upon injury and diminished in vitro organoid formation. Single-cell RNA sequencing uncovered interleukin-6 (IL-6) as being up-regulated upon damage, however only in young but not aging pituitary. Administering IL-6 to young mice promptly triggered pituitary stem cell proliferation, while blocking IL-6 or associated signaling pathways inhibited such reaction to damage. By contrast, IL-6 did not generate a pituitary stem cell activation response in aging mice, coinciding with elevated basal IL-6 levels and raised inflammatory state in the aging gland (inflammaging). Intriguingly, in vitro stem cell activation by IL-6 was discerned in organoid culture not only from young but also from aging pituitary, indicating that the aging gland’s stem cells retain intrinsic activatability in vivo, likely impeded by the prevailing inflammatory tissue milieu. Importantly, IL-6 supplementation strongly enhanced the growth capability of pituitary stem cell organoids, thereby expanding their potential as an experimental model. Our study identifies IL-6 as a pituitary stem cell activator upon local damage, a competence quenched at aging, concomitant with raised IL-6/inflammatory levels in the older gland. These insights may open the way to interfering with pituitary aging.

Gene inactivation of the orphan G protein‐coupled receptor LGR4, a paralogue of the epithelial‐stem‐cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo , LGR4‐deficient crypts or progenitors, but not LGR5‐deficient progenitors , die rapidly with marked downregulation of stem‐cell markers and Wnt target genes, including Lgr5 . Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy. Lgr4 , a paralogue of Lgr5 , is also expressed in intestinal crypts. Crypts from Lgr4 hypomorphic mice display signs of defective Wnt signalling: reduced cell proliferation and defective terminal differentiation of Paneth cells in vivo . When cultured ex vivo , the crypts show downregulation of stem cell markers, including Lgr5 , and die rapidly.

Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths worldwide, with its incidence continuing to rise. Regorafenib, a multi-kinase inhibitor approved for palliative treatment, has been shown to extend survival in patients with metastatic CRC (mCRC) who have failed standard therapies. However, its clinical benefit is limited to a subset of patients, is typically short-lived, and is often accompanied by significant toxicity. The mechanisms by which CRC cells develop resistance to regorafenib remain incompletely understood. In this study, we investigated the mechanisms of regorafenib resistance using a preclinical mouse colon organoid model. Transcriptomic analysis of Apc wild-type and Apc-deficient organoids treated with regorafenib revealed upregulation of epithelial-to-mesenchymal transition (EMT), alterations in the secretome, and increased activation of phosphorylated Erk1/2. Notably, co-treatment with an autophagy inhibitor suppressed regorafenib-induced EMT and its associated secretory phenotype, leading to reduced cell proliferation and enhanced apoptosis in mouse organoids. The efficacy of this drug combination was further supported by cell viability assays in human CRC cell lines. In contrast, primary mouse colon fibroblasts exhibited greater resistance to both single-agent and combination regorafenib treatments. In summary, our findings using an organoid model suggest that autophagy inhibition may represent a promising strategy to overcome chemoresistance to regorafenib in mCRC patients.

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.

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the \"pro-hair cell\" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

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.

A murine experimental model system aimed at developing potential vaccines to papovavirus infection in immunosuppressed individuals was explored. A VP1-pseudocapsid based on the major capsid protein of the murine polyomavirus A2 strain and a mutant, M17-pseudocapsid as well as four temperature sensitive ( ts)-mutants were used as immunogens. T-cells deficient CD4 −/−8 −/− mice were immunized four times with each immunogen and then together with non-immunized control mice challenged with polyomavirus. In contrast to all control mice, only half of the immunized mice exhibited presence of polyoma DNA when assayed by PCR. The results indicate that pseudocapsids and ts-mutant immunization may potentially protect mice with an impaired T-cell function from polyomavirus infection.

Cell plasticity governs tissue regeneration but can also drive metaplasia, the replacement of one cell type with another. This process increases the risk of cancer development in several tissues, including esophagus. Esophageal metaplasia development partly depends on keratinocyte plasticity, making regulation of esophageal progenitor fate critical. We previously identified Sox9 as instrumental in regulating esophageal cell plasticity following Hedgehog pathway activation. Our current study reveals that Hedgehog indirectly regulates Sox9 by modifying epithelial-stromal communication. This activates TGF-β and BMP pathways in epithelial cells, which synergistically regulate Sox9 and stimulate a transcriptomic program resembling squamo-columnar junction progenitors, which are prone to initiate metaplasia. Importantly, we demonstrate pharmacological modulation of this plasticity in vivo. Indeed, Ibuprofen inhibits Hedgehog-induced Sox9 expression by directly targeting epithelial cells, providing proof of concept for pharmacological intervention in cell plasticity with implications for regenerative medicine and metaplasia treatment.

Collagenase/dispase treatment of intestinal tissue from adult mice generates cells growing in matrigel as stably replatable cystic spheroids in addition to differentiated organoids. Contrary to classical EDTA-derived organoids, these spheroids display poor intestinal differentiation and are independent of Rspondin/Noggin/EGF for growth. Their transcriptome resembles strikingly that of fetal intestinal spheroids, with downregulation of crypt base columnar cell (CBC) markers (Lgr5, Ascl2, Smoc2, Olfm4). In addition, they display upregulation of inflammatory and mesenchymal genetic programs, together with robust expression of YAP target genes. Lineage tracing, cell-sorting and single cell RNA sequencing experiments demonstrate that adult spheroid-generating cells belong to a hitherto undescribed developmental lineage, independent of Lgr5+ve CBCs, and are involved in regeneration of the epithelium following CBC ablation. biorxiv;2024.03.11.584399v3/UFIG1F1ufig1

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Though MASH is closely tied to metabolic risk factors, the underlying pathogenic mechanisms remain scarcely understood. Recent research underscores the importance of the gut-liver axis in its pathogenesis, an aspect less explored in human studies. Here, we investigated whether the duodenal epithelium of MASH patients, could exhibit intrinsic dysfunctions. Duodenal epithelial organoids were generated from 16 MASH patients and 14 healthy controls. Biopsies and patient-derived organoid transcriptomes were then analyzed to evaluate if specific intestinal pathways were differentially modulated in MASH subjects. Functional assays were performed to assess the duodenal epithelial digestive potential and barrier functionality. Organoid formation efficiency was similar between control-derived epithelial organoids (CDEOs) and MASH-derived epithelial organoids (MDEOs) (71% and 69%, respectively). Despite global heterogeneity in growth patterns, MDEOs frequently exhibited cystic spheroid morphology. MDEOs displayed altered digestive homeostasis associated with reduced mature absorptive cell fate, but they retained their lipid metabolic capacity, possibly mediated by lipid oxidation in stem/progenitor cells. Additionally, MDEOs misexpressed components of tight and adherens junctions and desmosomes compared to controls. However, MDEOs maintained pore and leak pathway integrity, indicating that the duodenal epithelial barrier remained functionally preserved under tested conditions. This study provides evidence that the duodenal epithelium of MASH patients exhibits significant alterations in its digestive and barrier functions. This study sheds light on the intricate dynamics of duodenal epithelial alterations in MASH, highlighting potential therapeutic avenues for restoring intestinal homeostasis.