Prostaglandins produced by endometrial and conceptus tissues have an established role in maintaining luteal function in early pregnancy and initiating luteolysis during diestrus in mares. The cellular mechanisms controlling the secretion and delivery of prostaglandins have not been fully elucidated. We hypothesized that nanosized lipid bilayer extracellular vesicles (EV) that carry a variety of biological molecules would be involved in these key regulatory processes. Serum and uterine fluid samples were collected from diestrus and pregnant mares on day (D) 10 and D14 post-ovulation, and conceptuses were collected in pregnant mare cycles and cultured for 2–3 h. Extracellular vesicles were isolated using membrane affinity spin columns. Metabolomic EV cargo was assessed using LC–MS/MS, and cytosolic phospholipase A2 (cPLA2) with immunoblotting. Serum and uterine fluid PGEM and conceptus PGE2 concentrations were compared using ELISA. Prostaglandins were below detection limits in all EV serum and uterine fluid eluates. PGEM concentrations in uterine fluid and serum did not differ between pregnant and diestrus mares at these time points. Day 10 and D14 conceptuses produced PGE2. Serum and uterine fluid EV contained AA and cPLA2. While AA abundance did not differ by reproductive status or day at these time points, uterine fluid EV AA abundance was 83 times higher than serum. Serum and uterine fluid EV carry activatable phospholipases and rate-limiting substrates for prostaglandin production rather than the prostaglandins themselves. High levels of AA in uterine fluid EV are likely important in the local regulation of uterine prostaglandin production.
Mesenchymal stem cells (MSCs) possess significant metabolic plasticity, enabling them to adapt dynamically to environmental stressors during tissue repair. Although UCP1 is associated with nonshivering thermogenesis in brown adipose tissue (BAT) through the uncoupling of oxidative phosphorylation, its impact on MSCs metabolism remains unexplored. Herein, we evaluated the ectopic expression of UCP1 in MSCs and its effects on classical surface markers, the redox state and glucose metabolism. MSCs were genetically modified to constitutively overexpress UCP1 using a lentiviral construct. Transduction efficiency was evaluated by direct UCP1 expression and the co-expressed EGFP fluorescent tag. Surface marker phenotype and proliferation were evaluated by expression of multipotency markers and metabolic activity, respectively. Metabolic phenotypes were assessed by measuring mitochondrial membrane potential (ΔΨm), quantifying cytoplasmic and mitochondrial reactive oxygen species (ROS), and evaluating glucose uptake via the 2-NBDG assay, while key glycolytic markers were evaluated by qPCR and immunoblot. UCP1-overexpressing MSCs showed a marked reduction in ΔΨm; however, normalized intracellular ATP levels remained stable across varying cell densities. Crucially, persistent UCP1 expression did not affect surface markers or cell metabolic activity, retaining their classical surface marker profile; furthermore, the engineered cells showed reduced baseline ROS levels compared with the parental line. Most notably, MSC-UCP1 cells showed increased glucose uptake and increased sensitivity to glucose deprivation, displaying increased expression of glycolytic markers. Our data indicate that constitutive UCP1 expression is associated with a shift in MSC metabolism towards a glucose-dependent state. This distinct metabolic profile highlights the plasticity of MSCs energetics and suggests a potential therapeutic application for genetically engineered MSCs in treating energy imbalance disorders such as obesity and type 2 diabetes.
Reliable assessment of mesenchymal stromal cell (MSC) viability and stem cell–associated properties is essential for ensuring the consistency, safety, and therapeutic potential of MSC cultures before clinical use. However, incomplete understanding of the mechanisms underlying MSC therapeutic effects remains a major barrier to clinical translation and to establishing reliable, standardised quality attributes. Artificial intelligence (AI) may enable automated assessment of these properties, but the performance and clinical relevance of AI-based approaches have not been systematically evaluated. This systematic review evaluated the accuracy and clinical utility of AI for assessing viability and stem cell–associated properties in human MSC cultures. Following PRISMA guidelines, we searched five electronic databases (Cochrane, Scopus, PubMed, EMBASE, and MEDLINE) for eligible studies reporting quantitative AI-based assessments of human MSC cultures. Two reviewers independently screened studies, extracted data, and assessed risk of bias using PROBAST. The review protocol was prospectively registered with PROSPERO (CRD420256393235). Of 3,872 records identified, 18 studies met the inclusion criteria. Supervised AI models, particularly convolutional neural networks, were most used. Across included studies, reported performance metrics varied substantially because of differences in AI models, imaging modalities, datasets, and outcome definitions. Considerable heterogeneity in AI model architectures, imaging modalities, outcome definitions, and reporting metrics limited direct comparison across studies. Overall, available evidence suggests that AI can assess key MSC quality attributes and may support more objective, scalable, and standardised quality-control workflows. However, broader validation, standardised reporting, external testing, and evaluation across a wider range of MSC characteristics are required before routine clinical implementation can be recommended.
Regeneration in many animals involves the formation of blastema, a mass of undifferentiated, proliferating cells. The origin of blastema cells is a key question in regenerative biology. Two major theories are: (1) the migration of lineage-restricted stem cells, supported by studies in salamanders and planaria, and (2) the dedifferentiation of somatic cells at the injury site, which has been observed in newts, mice, zebrafish, and crickets. Because different models support these theories, the generality of either mechanism is unclear. To address this, these theories should be investigated in a wider range of regenerative model animals. To this end, Fujita et al. (2024) studied the regenerative annelid Enchytraeus japonensis and discovered that soxC-expressing cells are crucial for blastema formation. In this review, we introduce an annelid model system for regeneration biology and the roles of soxC-expressing cells during blastema formation.
In seahorses (family Syngnathidae), the male brood pouch provides a protected environment for developing embryos. During pregnancy, embryos develop within an enclosed brood pouch, requiring a physiological mechanism to eliminate the nitrogenous waste excreted by the embryos. This study investigated the expression and localization of rhesus (Rh) glycoproteins, expected to function as ammonia transporters, in the brood pouch of the pot-bellied seahorse Hippocampus abdominalis. The expression of the RhAG, RhBG, and RhCG2 genes in the pouch was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). In situ hybridization and immunohistochemistry revealed that RhBG and RhCG2 are localized in the inner epithelium of the pouch, while RhAG is present in erythrocytes and the endothelium of the blood vessels. Based on these findings, we propose the following waste removal pathway. Ammonia, presumably released from the embryo, would be transported from the lumen of the pouch through apical RhCG2 and basolateral RhBG of inner epithelial cells into the connective tissue beneath the inner epithelium. In the connective tissue, ammonia passes through RhAG-expressing endothelial cells into the vascular lumen and is likely taken up by RhAG-expressing erythrocytes, which then travel to the gills via the bloodstream. Then, ammonia is released from the RhAG-expressing erythrocytes through RhAG-expressing pillar cells, basolateral RhBG- and apical RhCG2-expressingpavement cells, finally into the surrounding seawater. This study provides a first molecular and cellular basis for a proposed ammonia removal mechanism within the seahorse brood pouch, highlighting physiological adaptations associated with male pregnancy in syngnathid fish.
