James A Ankrum

The in vivo fate of mesenchymal stromal cells (MSCs), including their clearance, interaction with host tissues, and persistence, remains incompletely understood following systemic or local clinical administration to patients. Although immune-mediated clearance mechanisms, such as triggering of the instant blood-mediated inflammatory reaction (IBMIR), coagulation and complement pathways activation, apoptosis, and efferocytosis have been identified, their contributions to MSC function and efficacy are still under investigation. To address these knowledge gaps, an international panel of global experts in MSC biology and clinical regenerative medicine convened to assess current evidence and define key unanswered questions. Discussions were structured around three thematic domains: (1) biodistribution and mechanisms following systemic delivery; (2) biological implications of local or depot-based administration, and (3) the dynamics of MSC persistence and clearance in vivo. A major focus was on the role of MSC apoptosis and its immunological consequences, particularly interactions between apoptotic MSCs, phagocytes, and endothelial barriers. This perspective highlights the most urgent research questions identified during the meeting and in follow-up discussions and proposes experimental strategies to move beyond traditional cell tracking toward interrogating functional persistence, immune modulation, and delivery context. Addressing these gaps will deepen our understanding of MSCs in vivo and guide the development of safer, predictable, and effective MSC-based interventions.

Type 1 diabetes (T1D) is caused by the selective autoimmune ablation of pancreatic β-cells. Emerging evidence reveals β-cell secretory dysfunction arises early in T1D development and may contribute to diseases etiology; however, the underlying mechanisms are not well understood. Our data reveal that proinflammatory cytokines elicit a complex change in the β-cell's Golgi structure and function. The structural modifications include Golgi compaction and loss of the interconnecting ribbon resulting in Golgi fragmentation. We further show that Golgi structural alterations coincide with persistent altered cell surface glycoprotein composition. Our data demonstrate that inducible nitric oxide synthase (iNOS)-generated nitric oxide (NO) is necessary and sufficient for β-cell Golgi restructuring. Moreover, the unique sensitivity of the β-cell to NO-dependent mitochondrial inhibition results in β-cell-specific Golgi alterations that are absent in other cell types, including α-cells. Examination of human pancreas samples from autoantibody-positive and T1D donors with residual β-cells further revealed alterations in β-cell, but not α-cell, Golgi structure that correlate with T1D progression. Collectively, our studies provide critical clues as to how β-cell secretory functions are specifically impacted by cytokines and NO that may contribute to the development of β-cell autoantigens relevant to T1D.
Proinflammatory cytokines drive disruptions in Golgi structure and function in human, mouse, and rat β-cells. Golgi alterations result from inducible nitric oxide synthase (iNOS)- and nitric oxide (NO)-dependent inhibition of mitochondrial metabolism. α-Cell Golgi structure is insensitive to cytokine- and NO-mediated metabolic inhibition. Analysis of human donor tissue shows early Golgi alteration in β-cells from autoantibody-positive donors, which persists in residual β-cells from T1D donors.

The ovarian follicle is the functional unit of the ovary that produces hormones and gametes needed to sustain female reproductive function and health. The ability to recapitulate folliculogenesis, ovulation, and luteinization in vitro has broad basic, translational, and clinical utility. The most advanced in vitro follicle growth systems maintain the follicle's three-dimensional (3D) architecture, which is crucial for the development of meiotically competent metaphase II oocytes in humans. Recently, a scaffold-free method for in vitro follicle growth of mouse multilayer secondary follicles was developed and validated. For this, custom 3D printed molds were used to micropattern agarose with microwells that accommodate the volumetric expansion of follicles. Follicles grown in this scaffold-free environment showed comparable hormone production and viability relative to well-established alginate-based encapsulated in vitro follicle growth (eIVFG) systems. Importantly, agarose microwells are a scalable method, less technically demanding, and show improved follicle growth and ovulation rates relative to eIVFG. This methodology produces customizable molds that are biocompatible with the oocyte, a cell highly sensitive to material-specific leachates and other environmental contaminants. Further, follicles in this system are cultured in the same focal plane, enabling real-time timelapse imaging and analysis. To increase the accessibility of this new approach, this article details the methods needed to design and 3D-print master molds, create silicone molds for 24- or 96-well plates, and culture isolated multilayer secondary ovarian follicles in the agarose molds. This setup can also be integrated with a cost-effective time-lapse imaging system, enabling morphokinetic analysis. In addition, molds can be paraffin-embedded for downstream histological analyses. Overall, this user-friendly method is a versatile tool for follicle culture and can be customized further to promote the differentiation and maturation of germ cells within the context of the follicle to sustain complete in vitro gametogenesis.

The immunomodulatory function of human mesenchymal stromal cells (MSCs) strongly depends on external factors; such as cytokines and other signalling molecules encountered in the disease microenvironment. An insufficiently inflammatory environment can fail to activate MSCs, and certain signals can impair their function. Obesity is on the rise worldwide, making it an additional factor to be considered prior to MSC therapy, as the microenvironment presents its own challenges. Elevated levels of serum free fatty acids, specifically palmitate, have the potential to affect MSC therapy. Palmitate-exposure has been shown to impair MSC immunomodulation of T cells in vitro. However, this is yet to be studied in the context of macrophages.
MSCs from three independent donors were exposed to 0.4mM of palmitate for 6-24 h. Gene expression, protein production and functional capacity were then assessed in response to palmitate. A ceramide synthesis inhibitor (Fumonisin B1) and a CC-chemokine ligand 2 (CCL2)-neutralising antibody were further used to assess the impact of these components on palmitate-associated immunomodulation.
We demonstrated that palmitate-exposed MSCs have enhanced suppression of human monocyte-derived macrophage (MDM) production of tumour necrosis factor α (TNFα), in a CCL2-dependent manner. We further elucidated parts of the pathway, such as ceramide synthesis, through which palmitate promotes this enhanced immunomodulation of macrophages.
Palmitate-exposed MSCs show enhanced immunomodulation of human MDMs, through the ceramide/CCL2 axis in vitro.

Polychlorinated biphenyls (PCBs) remain an environmental health concern due to their persistence and ongoing release from legacy and emerging sources. 2,2',5,5'-Tetrachlorobiphenyl (PCB52), a PCB congener frequently detected in the environment and human blood, is oxidized to 2,2',5,5'-tetrachlorobiphenyl-4-ol (4-52). The neurotoxicity of this hydroxylated (OH-PCB) metabolite remains poorly characterized. In this study, we exposed 4-week-old male Sprague Dawley rats to 4-52 via a polymeric implant drug delivery system grafted in the subcutaneous cavity at 4-52 concentrations of 0%, 1%, 5%, and 10% in the implant (w/w) for 28 days. Metabolomic analyses were performed in the serum. RNA sequencing, immunofluorescence, and dopamine (DA) measurement with electrochemical detection were used to characterize the effects of 4-52 on the striatum and cerebellum, brain regions implicated in PCB neurotoxicity. Serum metabolomic analysis revealed disruptions in the "arginine biosynthesis" pathway following 4-52 exposure. Exposure to 4-52 caused moderate transcriptomic changes in pathways related to "oxidative phosphorylation" and "neuroactive ligand-receptor interactions." Immunofluorescence showed no significant alterations in microglial, astrocytic, or apoptotic biomarkers. In the medium dose group, the levels of the DA metabolite DOPAL (3,4-dihydroxyphenylacetaldehyde) were significantly reduced in the striatum. Subsequent multi-omics network analysis identified interactions among OH-PCBs, endogenous metabolites, and the transcriptome. For example, levels of glutamic acid, aspartic acid, choline, and glycerophosphocholine negatively correlated with 4-52 in the striatum. Expression levels of heat shock protein (HSP) family genes, Hsp90b1, Hspa8, and Hspa5, positively correlated with serum metabolites, including proline, 1-methylguanidine, and methionine sulfoxide. These findings identify novel biomarkers and targets of 4-52-induced neurotoxicity.
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•2,2′,5,5′-Tetrachlorobiphenyl-4-ol (4−52) affected the brain transcriptome•4-52 disrupted the serum metabolome, including arginine biosynthesis•4-52 altered dopamine metabolism in the striatum in a dose-dependent manner•Network analysis identified novel targets and biomarkers of 4-52 exposure

Polychlorinated biphenyls (PCBs) are a group of environmental toxicants associated with increased risk of diabetes, obesity, and metabolic syndrome. These metabolic disorders are characterized by systemic and local inflammation within adipose tissue, the primary site of PCB accumulation. These inflammatory changes arise when resident adipose tissue macrophages undergo phenotypic plasticity – switching from an anti-inflammatory to an inflammatory phenotype. Thus, we sought to assess whether PCB exposure drives macrophage phenotypic switching. We investigated how human monocyte-derived macrophages polarized toward an M1, M2a, or M2c phenotype were impacted by exposure to Aroclor 1254, a PCB mixture found at high levels in school air. We showed that PCB exposure not only exacerbates the inflammatory phenotype of M1 macrophages but also shifts both M2a and M2c cells toward a more inflammatory phototype in both a dose- and time-dependent manner. Additionally, we show that PCB exposure leads to significant metabolic changes. M2 macrophages exposed to PCBs exhibit increased reliance on aerobic glycolysis and reduced capacity for fatty acid and amino acid oxidation—both indicators of an inflammatory macrophage phenotype. Collectively, these results demonstrate that PCBs promote immunometabolic macrophage plasticity toward a more M1-like phenotype, thereby suggesting that PCBs exacerbate metabolic diseases by altering the inflammatory environment in adipose tissue.

•Palmitate drives trained immunity in human macrophages dependent on epigenetic remodelling•Palmitate training promotes an M2 phenotypic switch with CD206 expression•MSCs suppress palmitate training pro-inflammatory cytokine production by macrophages•MSCs do not alter palmitate training induced M2 phenotypic switch•MSCs block palmitate training of macrophages via COX-2 and IL-1Ra
Innate training of macrophages can be beneficial for the clearance of pathogens. However, for certain chronic conditions innate training can have detrimental effects due to an excessive production of pro-inflammatory cytokines. Obesity is a condition that is associated with a range of increased pro-inflammatory training stimuli including the free fatty acid palmitate. Mesenchymal stromal cells (MSCs) are powerful immunomodulators and known to suppress inflammatory macrophages via a range of soluble factors. We show that palmitate training of murine bone-marrow derived macrophages and human monocyte-derived macrophages (MDMs) results in an increased production of TNFα and IL-6 upon stimulation with LPS and is associated with epigenetic remodelling. Palmitate training led to metabolic changes, however, MSCs did not alter the metabolic profile of human MDMs. Using a transwell system, we demonstrated that human bone marrow MSCs block palmitate training in both murine and human macrophages suggesting the involvement of secreted factors. MSC disruption of the training process occurs through more than one pathway. Suppression of palmitate enhanced TNFα production is associated with COX-2 activity in MSCs, while secretion of IL-1Ra by MSCs is required to suppress palmitate enhanced IL-6 production in MDMs.
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Polychlorinated biphenyls (PCBs) are linked to cancer, learning disabilities, liver and cardiovascular disease, and diabetes. Older schools often contain high levels of PCBs, and inhalation is a major source of exposure. Technical PCB mixtures, called Aroclors, and individual dioxin-like PCBs impair adipocyte function, which can lead to type II diabetes. To determine how PCB52, a non-dioxin like PCB congener found in school air, affects adipose, adolescent male and female rats were exposed to PCB52 by nose-only inhibition for 4h per day for 28 consecutive days. Transcriptomic analysis of white adipose revealed sex-specific differences in gene expression between PCB52- and sham-exposed males and females. Exposed females showed mitochondrial gene changes, including downregulation of the thermogenic uncoupling gene, Ucp1. Human preadipocytes/adipocytes exposed to PCB52 or its main metabolite, 4-OH-PCB52, also showed reduced norepinephrine-induced UCP1 expression. These findings suggest that PCB52 inhalation disrupts thermogenesis in adipose tissue, potentially contributing to metabolic syndrome.

Polychlorinated biphenyls (PCBs), such as PCB52, are hazardous environmental contaminants present in indoor and outdoor environments. Oral PCB exposure affects the colon microbiome; however, it is unknown if inhalation of PCBs alters the intestinal microbiome. We hypothesize that sub-acute inhalation of PCB52 affects microbial communities depending on the location in the (GI) gastrointestinal tract and the local profiles of PCB52 and its metabolites present in the GI tract following mucociliary clearance and biliary or intestinal excretion. Sprague-Dawley rats were exposed via nose-only inhalation 4hours per day, 7 days per week, for 4 weeks to either filtered air or PCB52. After 28 days, differences in the microbiome and levels of PCB52 and its metabolites were characterized throughout the GI tract. PCB52 inhalation altered taxa abundances and predicted functions altered throughout the gut, with most alterations occurring in the large intestine. PCB52 and metabolite levels varied across the GI tract, resulting in differing PCB × microbiome networks. Thus, the presence of different PCB52 and its metabolites in different parts of the GI tract has varying effects on the composition and predicted function of microbial communities. Future studies need to investigate whether these changes lead to adverse outcomes.
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•Inhalation of PCB 52 in rats alters the taxonomic composition in the cecum and colon.•Most predicted changes at the enzyme levels occur in the jejunum and ileum.•Bacterial alterations correlate with the level of PCB found in the intestinal content throughout the GI tract.

Introduction

Scarring from traumatic injury, burns, and other complications remains a significant problem that diminishes quality of life for millions of people worldwide. A common target for the development of new therapies to promote healing and reduce scarring are myofibroblasts because of their central role in pathological scarring. Recent work indicates that adipocyte lineage cells also contribute to the wound healing process, including clinical reports that indicate that the placement of autologous adipose micrografts at the surgical site improves the appearance and pliability of existing scars.
Methods

To better understand how adipocyte lineage cells interact with fibroblasts to promote healing, we first utilized an in vitro model of wound healing to visualize fibroblast spheroid collagen deposition via time-lapse imaging. We then introduced pre-adipocyte and adipocyte spheroids to visualize pair-wise spheroid interactions and collagen deposition among all three cell types. Finally, we quantified differences in the extracellular matrix (ECM) proteins produced using liquid chromatography with tandem mass spectrometry (LC-MS/MS).
Results

We found that all three cell-types contribute to ECM deposition and that the composition of the ECM proteins, or matrisome, was significantly different depending on which cells were co-cultured together.
Conclusions

By better understanding the interactions among these cell types, novel adipose-tissue-based therapeutic approaches can be developed to improve wound healing and reduce scar tissue.