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.

Abstract Description
 
We investigated whether exposure to polychlorinated biphenyls (PCBs) induces macrophage phenotypic switching toward an inflammatory phenotype. Human exposure to PCBs, an environmental toxicant, has been linked to an increased risk of diabetes, obesity, and metabolic syndrome. Despite the key role of adipose macrophages in metabolic disease and the known accumulation of PCBs in adipose, little is known about how PCBs impact macrophages. Here, we exposed anti-inflammatory macrophages (i.e. polarized with IL-4 or dexamethasone) to PCBs and then profiled phenotypic shifts by assessing surface marker expression, protein secretion, and energy metabolism. Our results show that PCB-exposed macrophages exhibited decreased expression of anti-inflammatory surface markers CD163 and CD206, while expression of inflammatory marker CD86 remained unchanged. PCB exposure also resulted in a marked 400x-1000x increase in the inflammatory cytokine IL-8 as well as a significant reduction in the anti-inflammatory cytokine IL-10. Additionally, we found that PCB-exposed cells had a greater dependence on aerobic glycolysis and a reduced ability to utilize fatty acid and amino acid oxidation as fuel – both metabolic features of more inflammatory macrophages. Collectively, these results demonstrate that PCBs promote macrophage plasticity toward a more inflammatory phenotype. More broadly, our work suggests that PCBs amplify metabolic diseases by altering the inflammatory environment of adipose tissue.

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.
[Display omitted]
•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

Interest in macrophages as a cell therapy is growing rapidly for the treatment of stroke, cardiomyopathy, and other indications. These therapies rely on in vitro polarization of macrophages before administration into the patient. While in vitro polarization toward an inflammatory or anti-inflammatory phenotype is effective, macrophages remain plastic and able to adapt to their environment upon transplantation. In this work, we aimed to uncover how in vitro polarized human macrophages would respond to exposure to a mixture of common environmental toxicants, Aroclor 1254. Aroclor 1254 is a mixture of polychlorinated biphenyls that were manufactured and used heavily in building materials and electrical equipment globally until its production was banned in the late 1970s. As a forever chemical, signatures of Aroclor 1254 are still commonly found in buildings, food, and people, where it can significantly alter biological processes.
Primary human monocytes were isolated from leukocyte reduction cones and purified using a monocyte negative selection kit. The monocytes were then differentiated into macrophages using M-CSF and then polarized to M1 (IFNg+LPS), M2a (IL-4), or M2c (Dexamethasone) macrophages. After in vitro polarization, each type of macrophage was challenged with exposure to either vehicle (DMSO) or 10 uM of Aroclor 1254 for 48 hours. Macrophages were then analyzed for changes to surface marker expression, cytokine production, and metabolic profile using a SCENITH assay.
Our results show that acute exposure of in vitro polarized macrophages to Aroclor 1254 significantly alter macrophage phenotype toward an inflammatory state. Specifically, levels of IL-10, CD163, and CD206 (anti-inflammatory markers) dropped significantly while production of inflammatory cytokines, such as IL-8, increased dramatically (Fig. 1A). When we analyzed the metabolic profile of macrophages after exposure to Aroclor 1254, we found they were more dependent on glycolytic processes (Fig. 1B) and less able to adapt to fatty acids or amino acid oxidation as a fuel source (Fig. 1C).
This work demonstrates that in vitro polarized macrophages are highly plastic and the signals they encounter post-transplantation can significantly alter their phenotype. In this work, we specifically showed that acute exposure to a common part of the exposome, polychlorinated biphenyls, can significantly alter the phenotype and secretome of macrophages by inducing an immunometabolic shift.

Adipose tissue is a potential source of cells for several types of cell therapy including MSCs, adipocytes, and macrophages. However, adipose tissue cellular composition and the function of the cells within adipose is heavily influenced by the metabolic health of the individual. With obesity rates reaching epidemic levels in the United States, it is important to understand how changes in metabolic state impact the function of adipose derived cells. To facilitate this work, we sought to create an in vitro model of adipose that incorporates adipocytes, MSCs, and macrophages. Our aim was to develop a co-culture technique to identify the influence of macrophage-adipocyte crosstalk on mature adipocyte function, adipose-derived MSC potential, and adipocyte influences on macrophage phenotype.
Stromal vascular fraction from surgical adipose tissue was culture expanded and characterized for MSC markers. MSC organoids were formed via agarose micromolds containing 35 microwells to form 10,000 cell organoids. The organoids were differentiated with adipogenic media for 14 days. Human monocytes were isolated from leukocyte reduction cones, differentiated into naïve macrophages, and polarized to either pro-inflammatory (M1-like) or anti-inflammatory (M2-like) states. To study mature adipocyte-macrophage cross-talk, micromolds were transferred to macrophage wells, cultured within agarose punchouts to control for macrophage seeding density and cell ratios. Media was collected for secretome analysis, and spheroids were stained and imaged through confocal microscopy for lipid content.
The secretome of macrophage/adipocyte co-cultures show distinct profiles between each cell type, and therefore, inflammatory environment. While both macrophage types caused diminished adiponectin production by adipocytes along with induced lipolysis without the presence of catecholamines, the presence of adipocytes lead to a decrease of pro-inflammatory cytokines with M2-like macrophages, yet an increase with M1s.
This work demonstrates the impact of adipose health on the quality of cells for cell therapies. Chronic inflammation impairs adipogenesis and impacts all the cells within adipose. Future studies need to be performed to identify the influence of inflammatory environment on MSC health, along with repeating further studies with additional donors.

The immunosuppressive activity of human mesenchymal stromal cells (MSCs) is central to the approved and investigational use of MSC therapy in inflammatory conditions such as GvHD. External factors like cytokines and other molecules encountered in the disease microenvironment significantly influence MSC immunosuppressive function. The obese microenvironment including elevated levels of serum free fatty acids, specifically palmitate, have the potential to affect MSC therapy. For T cell suppression, it has already been shown that exposure to palmitate impairs the ability of MSCs to carry out their function. However, we do not yet understand the effects of palmitate on MSC immunosuppression of macrophages. This study focused on investigating the influence of palmitate on MSC functional capacity to suppress macrophages and to elucidate the mechanisms of action at play.
We used peripheral blood from healthy control and patients with obesity to investigate the immunosuppressive capacity for MSCs or MSCs exposed to plamitate in vitro. Following exposure to palmitate, the capacity for MSCs to alter human monocyte derived macrophage (MDM) production of TNFa and IL-10 and phenotype following LPS stimulation were investigated. Moreover, chemical anatagonists, neutralising antibodies and a biologically active, cell-permeable ceramide analog were used to uncover the mechanisms of action.
Palmitate exposure significantly increased expression of the MSC immunomodulatory factors ptgs2, il-6, ccl2 and angptl4. Palmitate exposed MSCs had significantly modulated LPS stimulated human MDMs leading to decreased TNFα and increased IL-10 production by MDMs. Using a neutralising antibody, we identified that enhanced suppression mediated by palmitate exposed MSCs involved CCL2. Using the biologically active, cell-permeable ceramide analog C2 as well as the cermide synthases inhibitor (fumonisin B1), we showed that palmitate enhanced MSC immunomodulation of MDMs via activating ceramide de novo synthesis.
Palmitate has a beneficial effect on macrophage immunomodulation by MSCs, suggesting that a high concentration of palmitate in the microenvironment likely does not negatively affect MSC therapy in conditions where MSC -macrophage interactions are central to MSC mode of action.

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.