Mesenchymal stromal cells (MSCs) exert broad therapeutic effects across a range of inflammatory diseases. Their mechanism of action has largely been attributed to paracrine signalling, orchestrated by an array of factors produced by MSCs that are collectively termed the "secretome". Strategies to enhance the release of these soluble factors by pre-exposure to inflammatory cytokines, a concept known as "licensing", is thought to provide a means of enhancing MSC efficacy. Yet, recent evidence shows that intravenously infused MSCs entrapped within the lungs undergo apoptosis, and their subsequent clearance by host phagocytes is essential for their therapeutic efficacy. We therefore sought to clarify the mechanisms governing regulated cell death in MSCs and how exposure to inflammatory cytokines impacts this process. Our results show that MSCs are relatively resistant to cell death induced via the extrinsic pathway of apoptosis, as well as stimuli that induce necroptosis, a form of regulated inflammatory cell death. Instead, efficient killing of MSCs required triggering of the mitochondrial pathway of apoptosis, via inhibition of the pro-survival proteins MCL-1 and BCL-XL. Apoptotic bodies were readily released by MSCs during cell disassembly, a process that was inhibited in vitro and in vivo when the apoptotic effectors BAK and BAX were genetically deleted. Licensing of MSCs by pre-exposure to the inflammatory cytokines TNF and IFN-γ increased the sensitivity of MSCs to intrinsic apoptosis in vitro and accelerated their in vivo clearance by host cells within the lungs after intravenous infusion. Taken together, our study demonstrates that inflammatory "licensing" of MSCs facilitates cell death by increasing their sensitivity to triggers of the intrinsic pathway of apoptosis and accelerating the kinetics of apoptotic cell disassembly.
© 2025. The Author(s).

Deleterious accumulation of R-loops, a DNA-RNA hybrid structure, contributes to genome instability. They are associated with BRCA1 mutation-related breast cancer, an estrogen receptor α negative (ERα-) tumor type originating from luminal progenitor cells. However, a presumed causality of R-loops in tumorigenesis has not been established in vivo. Here, we overexpress mouse Rnaseh1 (Rh1-OE) in vivo to remove accumulated R-loops in Brca1-deficient mouse mammary epithelium (BKO). R-loop removal exacerbates DNA replication stress in proliferating BKO mammary epithelial cells, with little effect on homology-directed repair of double-strand breaks following ionizing radiation. Compared to their BKO counterparts, BKO-Rh1-OE mammary glands contain fewer luminal progenitor cells but more mature luminal cells. Despite a similar incidence of spontaneous mammary tumors in BKO and BKO-Rh1-OE mice, a significant percentage of BKO-Rh1-OE tumors express ERα and progesterone receptor. Our results suggest that rather than directly elevating the overall tumor incidence, R-loops influence the mammary tumor subtype by shaping the cell of origin for Brca1 tumors.

Hypertension (HTN) is a major cardiovascular disease that can cause and be worsened by renal damage and inflammation. We previously reported that renal lymphatic endothelial cells (LECs) increase in response to HTN and that augmenting lymphangiogenesis in the kidneys reduces blood pressure and renal pro-inflammatory immune cells in mice with various forms of HTN. Our aim was to evaluate the specific changes that renal LECs undergo in HTN.
We performed single-cell RNA sequencing. Using the angiotensin II-induced and salt-sensitive mouse models of HTN, we isolated renal CD31+ and podoplanin+ cells.
Sequencing of these cells revealed three distinct cell types with unique expression profiles, including LECs. The number and transcriptional diversity of LECs increased in samples from mice with HTN, as demonstrated by 597 differentially expressed genes (p < 0.01), 274 significantly enriched pathways (p < 0.01), and 331 regulons with specific enrichment in HTN LECs. These changes demonstrate a profound inflammatory response in renal LECs in HTN, leading to an increase in genes and pathways associated with inflammation-driven growth and immune checkpoint activity in LECs.
These results reinforce and help to further explain the benefits of renal LECs and lymphangiogenesis in HTN.
© 2024 The Author(s). Published by S. Karger AG, Basel.

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.

Endothelial cells are a heterogeneous population with various organ-specific and conserved functions that are critical to organ development, function, and regeneration. Here we report a Sox17-Erg direct reprogramming approach that uses cardiac fibroblasts to create differentiated endothelial cells that demonstrate endothelial-like molecular and physiological functions in vitro and in vivo. Injection of these induced endothelial cells into myocardial infarct sites after injury results in improved vascular perfusion of the scar region. Furthermore, we use genomic analyses to illustrate that Sox17-Erg reprogramming instructs cardiac fibroblasts toward an arterial-like identity. This results in a more efficient direct conversion of fibroblasts into endothelial-like cells when compared to traditional Etv2-based reprogramming. Overall, this Sox17-Erg direct reprogramming strategy offers a robust tool to generate endothelial cells both in vitro and in vivo, and has the potential to be used in repairing injured tissue.
© 2024. The Author(s).