Human Adipose Research Articles

3D bioprinting of fish skin-based gelatin methacryloyl (GelMA) bio-ink for use as a potential skin substitute.

Gelatin methacryloyl (GelMA), typically derived from mammalian sources, has recently emerged as an ideal bio-ink for three-dimensional (3D) bioprinting. Herein, we developed a fish skin-based GelMA bio-ink for the fabrication of a 3D GelMA skin substitute with a 3D bioprinter. Several concentrations of methacrylic acid anhydride were used to fabricate GelMA, in which their physical-mechanical properties were assessed. This fish skin-based GelMA bio-ink was loaded with human adipose tissue-derived mesenchymal stromal cells (ASCs) and human platelet lysate (HPL) and then printed to obtain 3D ASCs + HPL-loaded GelMA scaffolds. Cell viability test and a preliminary investigation of its effectiveness in promoting wound closure were evaluated in a critical-sized full thickness skin defect in a rat model. The cell viability results showed that the number of ASCs increased significantly within the 3D GelMA hydrogel scaffold, indicating its biocompatibility property. In vivo results demonstrated that ASCs + HPL-loaded GelMA scaffolds could delay wound contraction, markedly enhanced collagen deposition, and promoted the formation of new blood vessels, especially at the wound edge, compared to the untreated group. Therefore, this newly fish skin-based GelMA bio-ink developed in this study has the potential to be utilized for the printing of 3D GelMA skin substitutes.

7877 Adipose Depot Differences In Regenerative Properties Of Exosomes Derived From Human Adipose Stem Cells

Abstract Disclosure: M.A. Krause-Hauch: None. R. Patel: None. B. Osborne: None. P. Albear: None. N.A. Patel: None. Many patients struggle with chronic recalcitrant wounds that are the result of delayed healing. Underlying inflammation is one major risk factor for impaired dermal wound healing that may lead to pathologies such as infection and scarring. Adult human adipose stem cells (hASCs) have shown tremendous potential in regenerative medicine. The regenerative potential of hASCs is dependent on secreted bioactive material which is packaged and released in extracellular exosomes. We previously showed that hASC exosomes from the subcutaneous depot (sc) promoted repair in human dermal fibroblasts using in vitro assays. In this study, we evaluated exosomes derived from the omental depot (om) of hASC and compared its efficacy with sc-hASC. Our results show differences in levels of the long noncoding RNA (lncRNA) cargo between om-hASCexo and sc-hASCexo. Next, we evaluated their repair potential in a wound model in vivo. Male and female F344 rats were dorsally wounded via punch biopsy and silicone scaffolding was attached to prevent contraction. The wounds were then either not treated (control) or treated with 50μg om-hASCexo or sc-hASCexo every alternate day when each wound size was measured for 1 week to monitor healing progress. After 7 days, wounds treated with either om-hASCexo and sc-hASCexo closed significantly faster than untreated control wounds. While no difference was observed in closure percentage after 7 days for wounds treated with either om-hASCexo and sc-hASCexo, sc-hASCexo appeared to act faster earlier (1-2 days post wound) in the wound healing process while om-hASCexo acts faster during later stages (2-4 days post wound) of wound healing. The wound tissue was collected for biochemical and histological analysis. qPCR analysis on day 7 revealed that both om-hASCexo and sc-hASCexo treatment resulted in increased levels of Collagen 1 & 3 and MMP9, while levels of TGF-β decreased with treatment. These results indicate that om-hASCexo and sc-hASCexo enhanced the wound healing process while decreasing inflammation of the wound. To determine the differences between om-hASCexo and sc-hASCexo treatments on wound healing pathways, RNAseq was performed. Results show specificity in pathway activation and gene expression between the om-hASCexo and sc-hASCexo treatments. In conclusion, we have demonstrated that the regenerative mechanisms differ between the hASC depots from which exosomes are secreted. At a broader level, this finding may shed light on the body’s organ health and repair processes in normal or disease states. Presentation: 6/3/2024

6711 Single Nuclei RNA-seq To Map Adipose Cellular Populations And Senescent Cells In Response To A Lifestyle Or Senolytic Intervention In Older Adults With Obesity: A Double-Blind, Placebo-Controlled, Randomized Trial

Abstract Disclosure: A. Aslamy: None. G. Connolly: None. J. Nie: None. S. Espinoza: None. M. Serra: None. N. Musi: None. It is well established that obese and older individuals are at significantly increased risk for diabetes and metabolic syndrome. However, the underlying molecular mechanisms by which increased adiposity and aging predisposes individuals to metabolic disease are not well understood. Recent data suggests that variations in adipocyte cellular populations and subpopulations influence whole body metabolism. Particularly, an increased burden of adipocyte senescent cells (cells that are in a state of replicative arrest) have been implicated in metabolic diseases. To investigate the relationship between adipose molecular signatures and cellular senescence on metabolic outcomes, we will use a novel, high-throughput, high-resolution methodology called single nuclei RNA sequencing (SnRNA-seq) to develop a transcriptomic atlas of human adipose tissue and analyze molecular profiles before and after interventions of lifestyle vs. senolytic vs. placebo. We will recruit a cohort of older obese participants (≥65 years of age; BMI = 30-39.9 kg/m2; n = 72), and conduct a double-blinded, placebo-controlled, randomized trial in which we obtain adipose biopsies to analyze cellular/molecular profiling via snRNA-seq as it relates to phenotypic measures of insulin sensitivity, glucose tolerance, beta cell function, and hepatic lipid content compared to cohorts of younger lean (18-30 years of age; BMI = 18.5-24.9 kg/m2; n = 24) and older lean (≥65 years of age; BMI = 18.5-24.9 kg/m2; n = 24) participants. Older obese participants will be randomized to one of three groups for a 10-week period: lifestyle (diet- and exercise-induced weight loss of 8-10% of body mass), senolytic (dasatinib and quercetin), or placebo. Repeat adipose biopsies and metabolic testing will be analyzed before and after intervention to assess potential changes in gene expression and associated phenotypic measures. Inclusion criteria for the older obese participants are the following: ≥65 years of age, BMI 30-39.9 kg/m2, any sex, race and ethnic group, sedentary (≤1.5h of exercise per week), and nondiabetic. Exclusion criteria include history of diabetes, participation in structured exercise >1.5h per week, chronic inflammatory/neurologic/autoimmune/pulmonary disorders, GI or heart disease, use of anti-arrhythmic or weight altering medications, smoking/alcohol use, previous bariatric surgery, and use of/allergy to senolytic agents. Preliminary snRNA-seq analyses of adipose biopsies from our pilot study with non-diabetic participants identified cells with elevated expression of positive regulators of senescence (CDKN1A, CDKN2A, ARG2); notably, one subpopulation of senescent adipocytes, termed NewA1, was increased with higher BMI. Overall, the findings from this study may help us gain better understanding of the aging process and identify potential molecular targets for prevention of metabolic disease. Presentation: 6/1/2024

Prunella vulgaris and Tussilago farfara demonstrate anti-inflammatory activity in rabbits and protect human adipose stem cells against thermal stress in vitro

Ethnopharmacological relevancePrunella vulgaris L.(PV) and Tussilago farfara (TF) are perennial herbs rich in flavonoids and phenolic compounds with immense medicinal value. PV extract (PV-E) possesses potent antipyretic, anti-inflammtory, antioxidant, antiseptic, anti-cancer and immune stimulatory properties and have been traditionally known for the treatment of wounds, ulcers and sores. TF extract (TF-E) has been known for antibacterial, antioxidant, anti-inflammatory, anti-viral, anti-diabetic, anti-cancer, anti-obesity and wound healing effects. Additionally, TF-E infusions have been used for asthma, cough, and bronchopneumonia treatments. Aim of the studyThe therapeutic efficacy of transplanted human adipose stem cells (hASCs) is abrogated under the deteriorating effects of heat stress offered by burn wounds. Earlier researches has documented antioxidant priming as an effective strategy to enhance stem cell performance. As both PV-E and TF-E are known for their potent antioxidant effects. The present study aims to examine the cryoprotective effects of PV-E and TF-E priming on hASCs against in-vitro heat-induced thermal stress. Moreover, we determined the anti-inflammatory potential of both PV-E and TF-E on rabbits. MethodsAntioxidant capacity of both PV-E and TF-E is examined via DPPH assay and anti-inflammatory activity is assessed in rabbits using carrageen-induced paw edema model of inflammation. Next, we investigate the efficacy of different doses (1.25-100μg/ml) of PV-E and TF-E on hASCs; MTT, LDH, calcein AM staining, and wound scratch assay were used to assess cell viability, cytotoxicity, proliferation ability and cell migration potential in the cells. Then, hASCs were pretreated for 24 h with optimum doses of PV-E and TF-E determined from MTT assay results and were subsequently exposed to in-vitro thermal injury (51 °C,10 min). The cytoprotective effects of both PV-E and TF-E priming under thermal stress were investigated via MTT, LDH, annexin-V staining and gene expression analysis. ResultsBoth PV-E and TF-E extracts demonstrated potent antioxidant and effective anti-inflammatory activities, with a clear reduction in inflammation. Study on hASCs exhibited improved cell viabilities, enhanced cell proliferation and migration abilities of both extracts. While heat stress data revealed that PV-E (2.5μg/ml) and TF-E (5μg/ml) pretreatment significantly ameliorated effects of thermal-injuries in hASCs as depicted by significantly enhanced cell viabilities, low LDH release profile, and lower annexin-V expression and regulated gene expression of the pretreated cells. ConclusionPV-E and TF-E priming effectively enabled hASCs to combat thermal injury by significantly promoting cell survival than untreated cells. Hence, these findings suggest that PV-E and TF-E priming could be used to attain improved cellular responses and enhanced therapeutic efficacy in burnt tissue.

Efficacy and Safety of Cell-Assisted Acellular Adipose Matrix Transfer for Volume Retention and Regeneration Compared to Hyaluronic Acid Filler Injection.

Cell-assisted acellular adipose matrix (AAM) transfer is a novel technique for soft tissue volume restoration, where AAM acts as a scaffold for tissue proliferation and promotes host cell migration, vascularization, and adipogenesis. This study aimed to evaluate the efficacy and safety of in vivo cell-assisted AAM transfer compared to hyaluronic acid (HA) filler injection. Human adipose tissue was used to manufacture AAM, and murine adipose-derived stem cells (ASCs) were prepared. Nude mice were divided into four groups: AAM transfer (AT), ASC-assisted AAM transfer (CAT), HA filler injection (HI), and ASC-assisted HA filler injection (CHI). Eight weeks post-transfer, in vivo graft volume/weight, histology, and gene expression were analyzed to assess efficacy and safety. The AAM retained its three-dimensional scaffold structure without cellular components. AT/CAT showed lower volume retention than HA/CHA; however, CAT maintained a similar volume to HA. Histologically, adipogenesis and collagen formation were increased in AT/CAT compared to HA/CHA, with CAT showing the highest levels. CAT also demonstrated superior angiogenesis, adipogenesis, and gene expression (Vegf and Pparg), along with lower Il-6 expression, higher Il-10 expression, and reduced capsule formation, indicating better biocompatibility. Cell-assisted AAM transfer is a promising technique for volume retention and tissue regeneration, offering a safe and effective alternative to HA filler injections. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

In Vitro Treatment with Metformin Significantly Reduces Senescent B Cells Present in the Adipose Tissue of People with Obesity

BackgroundOur previous work has shown that senescent B cells accumulate in the human adipose tissue (AT) from people with obesity, where they express transcripts for markers associated with the senescence-associated secretory phenotype (SASP) and secrete multiple inflammatory mediators. These functions of AT-derived B cells are metabolically supported. ObjectivesTo show that Metformin (MET), a widely used hypoglycemic and antidiabetic drug, is able at least in vitro to decrease frequencies, secretory profile, and metabolic requirements of senescent B cells isolated from the AT from people with obesity. MethodsWe recruited adult females with obesity (n = 8, age 40 ± 2 y, BMI range: 33–42) undergoing breast reduction surgery, who donated their discarded subcutaneous AT. B cells from stromal vascular fractions isolated after collagenase digestion of the AT were evaluated after in vitro incubation with MET (1 mM × 106 B cells) or with a medium for the following measures. The expression of transcripts for SASP-associated markers (p16INK4a and p21CIP1/WAF1) measured by quantitative polymerase chain reaction (qPCR); secretion of inflammatory cytokines (TNF-α, IL-6, IFN-γ and IL-17A) measured by a Cytometric Bead Array); metabolic characteristics as identified by a glycolytic test and Seahorse technology, and by the expression of transcripts for glucose transporters and metabolic enzymes involved in glucose metabolic pathways, measured by qPCR. To examine differences between MET-treated compared with untreated groups, paired Student’s t tests (two-tailed) were employed. ResultsMET in vitro was able to reduce frequencies and numbers of senescent B cells, as identified by staining with β-galactosidase, as well as the secretion of inflammatory cytokines, the expression of transcripts for SASP, and metabolic markers that support intrinsic B cell inflammation. ConclusionsOur results provide evidence to support the beneficial effects of MET in reducing AT-related inflammation through its effects on senescent B cells.

A novel long non-coding RNA connects obesity to impaired adipocyte function

BackgroundLong non-coding RNAs (lncRNAs) can perform tasks of key relevance in fat cells, contributing, when defective, to the burden of obesity and its sequelae. Here, scrutiny of adipose tissue transcriptomes before and after bariatric surgery (GSE53378) granted identification of 496 lncRNAs linked to the obese phenotype. Only expression of linc-GALNTL6-4 displayed an average recovery over 2-fold and FDR-adjusted p-value <0.0001 after weight loss. The aim of the present study was to investigate the impact on adipocyte function and potential clinical value of impaired adipose linc-GALNTL6-4 in obese subjects. MethodsWe employed transcriptomic analysis of public dataset GSE199063, and cross validations in two large transversal cohorts to report evidence of a previously unknown association of adipose linc-GALNTL6-4 with obesity. We then performed functional analyses in human adipocyte cultures, genome-wide transcriptomics, and untargeted lipidomics in cell models of loss and gain of function to explore the molecular implications of its associations with obesity and weight loss. ResultsThe expression of linc-GALNTL6-4 in human adipose tissue is adipocyte-specific and co-segregates with obesity, being normalized upon weight loss. This co-segregation is demonstrated in two longitudinal weight loss studies and two cross-sectional samples. While compromised expression of linc-GALNTL6-4 in obese subjects is primarily due to the inflammatory component in the context of obesity, adipogenesis requires the transcriptional upregulation of linc-GALNTL6-4, the expression of which reaches an apex in terminally differentiated adipocytes. Functionally, we demonstrated that the knockdown of linc-GALNTL6-4 impairs adipogenesis, induces alterations in the lipidome, and leads to the downregulation of genes related to cell cycle, while propelling in adipocytes inflammation, impaired fatty acid metabolism, and altered gene expression patterns, including that of apolipoprotein C1 (APOC1). Conversely, the genetic gain of linc-GALNTL6-4 ameliorated differentiation and adipocyte phenotype, putatively by constraining APOC1, also contributing to the metabolism of triglycerides in adipose. ConclusionsCurrent data unveil the unforeseen connection of adipocyte-specific linc-GALNTL6-4 as a modulator of lipid homeostasis challenged by excessive body weight and meta-inflammation.

Dipeptidyl peptidase-4 marks distinct subtypes of human adipose stromal/stem cells with different hepatocyte differentiation and immunoregulatory properties

BackgroundHuman adipose-derived stromal/stem cells (hASCs) play important roles in regenerative medicine and numerous inflammatory diseases. However, their cellular heterogeneity limits the effectiveness of treatment. Understanding the distinct subtypes of hASCs and their phenotypic implications will enable the selection of appropriate subpopulations for targeted approaches in regenerative medicine or inflammatory diseases.MethodshASC subtypes expressing dipeptidyl peptidase-4 (DPP4) were identified via fluorescence-activated cell sorting (FACS) analysis. DPP4 expression was knocked down in DPP4+ hASCs via DPP4 siRNA. The capacity for proliferation, hepatocyte differentiation, inflammatory factor secretion and T-cell functionality regulation of hASCs from DPP4−, DPP4+, and control siRNA-treated DPP4+ hASCs and DPP4 siRNA-treated DPP4+ hASCs were assessed.ResultsDPP4+ hASCs and control siRNA-treated DPP4+ hASCs presented a lower proliferative capacity but greater hepatocyte differentiation capacity than DPP4− hASCs and DPP4 siRNA-treated DPP4+ hASCs. Both DPP4+ hASCs and DPP4− hASCs secreted high levels of vascular endothelial growth factor-A (VEGF-A), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6), whereas the levels of other factors, including matrix metalloproteinase (MMP)-1, eotaxin-3, fractalkine (FKN, CX3CL1), growth-related oncogene-alpha (GRO-alpha, CXCL1), monokine induced by interferon-gamma (MIG), macrophage inflammatory protein (MIP)-1beta, and macrophage colony-stimulating factor (M-CSF), were significantly greater in the supernatants of DPP4+ hASCs than in those of DPP4− hASCs. Exposure to hASC subtypes and their conditioned media triggered changes in the secreted cytokine profiles of T cells from healthy donors. The percentage of functional T cells that secreted factors such as MIP-1beta and IL-8 increased when these cells were cocultured with DPP4+ hASCs. The percentage of polyfunctional CD8+ T cells that secreted multiple factors, such as IL-17A, tumour necrosis factor alpha (TNF-α) and TNF-β, decreased when these cells were cocultured with supernatants derived from DPP4+ hASCs.ConclusionsDPP4 may regulate proliferation, hepatocyte differentiation, inflammatory cytokine secretion and T-cell functionality of hASCs. These data provide a key foundation for understanding the important role of hASC subpopulations in the regulation of T cells, which may be helpful for future immune activation studies and allow them to be customized for clinical application.

The effect of exogenous mitochondria in enhancing the survival and volume retention of transplanted fat tissue in a nude mice model

BackgroundDespite the pivotal role of fat grafting in plastic, reconstructive, and aesthetic surgery, inconsistent survival rates of transplanted adipose tissue, primarily due to early ischemic and hypoxic insults, remain a significant challenge. The infusion of healthy mitochondria has emerged as a promising intervention to support tissue recovery from ischemic, hypoxic, and other types of damages across various organ systems.ObjectivesThis study aims to evaluate the impact of supplementing human adipose tissue grafts with healthy exogenous mitochondria on their volume and mass retention rates when transplanted into the subcutaneous layers of nude mice. This approach seeks to improve and optimize fat grafting techniques.MethodsHuman adipose tissues were preconditioned with exogenous mitochondria (10 µg/mL), a combination of exogenous mitochondria and the inhibitor Dyngo-4a, Dyngo-4a alone, or PBS, and then transplanted into the subcutaneous tissue of 24 nude mice. Samples were harvested at 1 and 3 months post-transplantation for analysis of mass and volume retention. The structural morphology and integrity of the adipose tissues were assessed using Hematoxylin and Eosin (H&E) staining.ResultsMitochondrial preconditioning significantly enhanced the retention of mass and volume in fat grafts, demonstrating superior structural morphology and integrity compared to the control group.ConclusionsThis study highlights the potential of exogenous mitochondrial augmentation in fat transplantation to significantly improve fat graft survival, thereby optimizing the success of fat grafting procedures.

Electrospun PCL Nerve Wrap Coated with Graphene Oxide Supports Axonal Growth in a Rat Sciatic Nerve Injury Model.

Background/Objectives: Peripheral nerve injuries (PNIs) are a debilitating problem, resulting in diminished quality of life due to the continued presence of both chronic and acute pain. The current standard of practice for the repair of PNIs larger than 10 mm is the use of autologous nerve grafts. Autologous nerve grafts have limitations that often result in outcomes that are not sufficient to remove motor and sensory impairments. Bio-mimetic nanocomposite scaffolds combined with mesenchymal stem cells (MSCs) represent a promising approach for PNIs. In this study, we investigated the potential of an electrospun wrap of polycaprolactone (PCL) + graphene oxide (GO), with and without xenogeneic human adipose tissue-derived MSCs (hADMSCs) to use as a platform for neural tissue engineering. Methods: We evaluated, in vitro and in vivo, the potential of the nerve wrap in providing support for axonal growth. To establish the rat sciatic nerve defect model, a 10 mm long limiting defect was created in the rat sciatic nerve of 18 Lewis rats. Rats treated with the nanocomposites were compared with autograft-treated defects. Gait, histological, and muscle analyses were performed after sacrifice at 12 weeks post-surgery. Results: Our findings demonstrate that hADMSCs had the potential to transdifferentiate into neural lineage and that the nanocomposite successfully delivered hADMSCs to the injury site. Histologically, we show that the PCL + GO nanocomposite with hADMSCs is comparable to the autologous nerve graft, to support and guide axonal growth. Conclusions: The novel PCL + GO nerve wrap and hADMSCs used in this study provide a foundation on which to build upon and generate future strategies for PNI repair.

Cytokine priming enhances the antifibrotic effects of human adipose derived mesenchymal stromal cells conditioned medium

BackgroundFibrosis is a pathological scarring process characterized by persistent myofibroblast activation with excessive accumulation of extracellular matrix (ECM). Fibrotic disorders represent an increasing burden of disease-associated morbidity and mortality worldwide for which there are limited therapeutic options. Reversing fibrosis requires the elimination of myofibroblasts, remodeling of the ECM, and regeneration of functional tissue. Multipotent mesenchymal stromal cells (MSC) have antifibrotic properties mediated by secreted factors present in their conditioned medium (MSC-CM). However, there are no standardized in vitro assays to predict the antifibrotic effects of human MSC. As a result, we lack evidence on the effect of cytokine priming on MSC’s antifibrotic effects. We hypothesize that the MSC-CM promotes fibrosis resolution in vitro and that this effect is enhanced following MSC cytokine priming.MethodsWe compared the antifibrotic effects of resting versus interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) primed MSC-CM in four in vitro assays: prevention of fibroblast activation, myofibroblasts deactivation, ECM degradation and fibrosis resolution in lung explant cultures. Furthermore, we performed transcriptomic analysis of myofibroblasts treated or not with resting or primed MSC-CM and proteomic characterization of resting and primed MSC-CM.ResultsWe isolated MSC from adipose tissue of 8 donors, generated MSC-CM and tested each MSC-CM independently. We report that MSC-CM treatment prevented TGF-β induced fibroblast activation to a similar extent as nintedanib but, in contrast to nintedanib, MSC-CM reduced fibrogenic myofibroblasts (i.e. transcriptomic upregulation of apoptosis, senescence, and inflammatory pathways). These effects were larger when primed rather than resting MSC-CM were used. Priming increased the ability of MSC-CM to remodel the ECM, reducing its content of collagen I and fibronectin, and reduced the fibrotic load in TGF-β treated lung explant cultures. Priming increased the following antifibrotic proteins in MSC-CM: DKK1, MMP-1, MMP-3, follistatin and cathepsin S. Inhibition of DKK1 reduced the antifibrotic effects of MSC-CM.ConclusionsIn vitro, MSC-CM promote fibrosis resolution, an effect enhanced following MSC cytokine priming. Specifically, MSC-CM reduces fibrogenic myofibroblasts through apoptosis, senescence, and by enhancing ECM degradation. Future studies will establish the in vivo relevance of MSC priming to fibrosis resolution.

An optimized pipeline for high-throughput bulk RNA-Seq deconvolution illustrates the impact of obesity and weight loss on cell composition of human adipose tissue.

Katie Whytock ( Katie.Whytock@adventhealth.com ).

GDF3 promotes adipose tissue macrophage-mediated inflammation via altered chromatin accessibility during aging.

Age-related susceptibility to sepsis and endotoxemia is poorly defined, although hyperactivation of the immune system and the expansion of the visceral adipose as an immunological reservoir are underlying features. Macrophages from older organisms exhibit substantial changes, including chronic NLRP3 inflammasome activation, genomic remodeling and a dysfunctional, amplified inflammatory response upon new exposure to pathogen. However, the mechanisms by which old macrophages maintain their inflammatory phenotype during endotoxemia remains elusive. We previously identified Gdf3 , a TGFβ superfamily cytokine, as a top-regulated gene by age and the NLRP3 inflammasome in adipose tissue macrophages (ATMs). Here, we demonstrate that endotoxemia increases inflammatory (CD11c + ) ATMs in a Gdf3- dependent manner in old mice. Lifelong systemic or myeloid-specific deletion of Gdf3 leads to reduced endotoxemia- induced inflammation, with decreased CD11c + ATMs and inflammatory cytokines, and protection from hypothermia. Moreover, acute blockade of Gdf3 using JQ1, a BRD4 inhibitor, phenocopies old mice with lifelong Gdf3- deficiency. We show that GDF3 promotes the inflammatory phenotype in ATMs by phosphorylating SMAD2/3. Mechanistically, the differential chromatin landscape of ATMs from old mice with or without myeloid-driven Gdf3 indicates that GDF3- SMAD2/3 signaling axis shifts the chromatin accessibility of ATMs towards an inflammatory state during aging. Furthermore, pharmaceutical inhibition of SMAD3 with a specific inhibitor of SMAD3 (SIS3) mimics Gdf3 deletion. SIS3 reduces endotoxemia-mediated inflammation with fewer CD11c + ATMs and less severe hypothermia in old, but not young mice, as well as reduced mortality. In human adipose tissue, age positively correlates with GDF3 level, while inflammation correlates with pSMAD2/3 level. Overall, these results highlight the importance of GDF3-SMAD2/3 axis in driving inflammation in older organisms and identify this signaling axis as a promising therapeutic target for mitigating endotoxemia-related inflammation in the aged.

Combining evidence from human genetic and functional screens to identify pathways altering obesity and fat distribution.

Overall adiposity and body fat distribution are heritable traits associated with altered risk of cardiometabolic disease and mortality. Performing rare variant (minor allele frequency<1%) association testing using exome-sequencing data from 402,375 participants in the UK Biobank (UKB) for nine overall and tissue-specific fat distribution traits, we identified 19 genes where putatively damaging rare variation associated with at least one trait (Bonferroni-adjusted P<1.58×10-7) and 52 additional genes at FDR≤1% (P≤4.37×10-5). These 71 genes exhibited higher (P=3.58×10-18) common variant prioritisation scores than genes not significantly enriched for rare putatively damaging variation, with evidence of monotonic allelic series (dose-response relationships) among ultra-rare variants (minor allele count≤10) in 22 genes. Five of the 71 genes have cognate protein UKB Olink data available; all five associated (P<3.80×10-6) with three or more analysed traits. Combining rare and common variation evidence, allelic series and proteomics, we selected 17 genes for CRISPR knockout in human white adipose tissue cell lines. In three previously uncharacterised target genes, knockout increased (two-sided t-test P<0.05) lipid accumulation, a cellular phenotype relevant for fat mass traits, compared to Cas9-empty negative controls: COL5A3 (fold change [FC]=1.72, P=0.0028), EXOC7 (FC=1.35, P=0.0096), and TRIP10 (FC=1.39, P=0.0157); furthermore, knockout of SLTM resulted in reduced lipid accumulation (FC=0.51, P=1.91×10-4). Integrating across population-based genetic and in vitro functional evidence, we highlight therapeutic avenues for altering obesity and body fat distribution by modulating lipid accumulation.

Therapeutic potential of mesenchymal stromal cells on morphological parameters in the hippocampus of rats with brain ischemia-reperfusion modeling

Ischemic stroke is an extremely important pathology with high mortality, in which more than 50 % of patients with occlusion of the main vessels remain disabled, despite early reperfusion therapy by thrombolysis or thrombectomy. As part of the regenerative strategy, stem cell transplantation in ischemic stroke became a new impetus. Cell therapy with the use of mesenchymal stromal cells demonstrated encouraging results regarding endogenous mechanisms of neuroregeneration in response to ischemic damage to brain structures. The aim of the research is to study the influence of mesenchymal stromal cells of various genesis, lysate of mesenchymal stromal cells obtained from Wharton’s jelly umbilical cord and citicoline on the dynamics of morphological changes in the hippocampal CA1 region of rats with acute cerebral ischemia-reperfusion according to light microscopy and micromorphometry data. The experiment was carried out using 200 male Wistar rats, which were subjected to ischemia-reperfusion by reversible 20-minute bilateral occlusion of the internal carotid arteries. Animals with modeled pathology were intravenously transplanted with mesenchymal stromal cells of various genesis (from Wharton’s jelly of the human umbilical cord, human and rat adipose tissue), and rat embryonic fibroblasts, lysate of mesenchymal stromal cells and citicoline were injected. Histological analysis of rat brain sections was performed on the 7th and 14th day of the experiment. Statistical analysis was performed using “Statistica 6.0” (StatSoft® Snc, USA). The significance of differences was assessed using the Student’s t-test and the nonparametric Mann-Whitney U test. During the study, it was found that modeled ischemia-reperfusion in rats caused almost complete degeneration of the structure of the pyramidal layer of hippocampal CA1 region, gave uniformity to the structure of the radiant layer, infiltration of microglia, contributed to the disruption of the arrangement of apical dendrite bundles and narrowing of blood vessels as a result of perivascular edema. Also, the modeled pathology reduced the total number of neuronal nuclei in the hippocampal CA1 area, the overwhelming majority of which had signs of pathological changes. Transplantation of mesenchymal stromal cells of various origins, lysate of mesenchymal stromal cells and citicoline contributed to a significant increase in the number of neuronal nuclei in the hippocampal CA1 zone and nuclei that did not undergo pathological changes. The most positive effect was found in the transplantation of mesenchymal stromal cells from human Wharton’s jelly-derived cells. Thus, both in the subacute and recovery periods of ischemic stroke in rats, the transplantation of human Wharton’s jelly-derived mesenchymal stromal cells was significantly surpassed the reference drug citicoline in its ability to reduce the number of pathologically changed nuclei by 1.5 times (p&lt;0.05). At the same time, the number of pathologically unchanged nuclei significantly exceeded the number of nuclei with signs of karyorrhexis and karyopyknosis, so it would be advisable to use mesenchymal stromal cells of various genesis, lysate or citicoline in conditions of acute cerebral ischemia-reperfusion, taking into account their ability to reduce the volume of the infarct. In the future, an injectable drug will be created from the most effective culture of mesenchymal stromal cells in terms of cerebroprotective properties for cell therapy of patients with acute ischemic stroke.

The adipose tissue melanocortin 3 receptor is targeted by ghrelin and leptin and may be a therapeutic target in obesity

ObjectiveObesity is linked to perturbations in energy balance mechanisms, including ghrelin and leptin actions at the hypothalamic circuitry of neuropeptide Y (NPY) and melanocortin. However, information about the regulation of this system in the periphery is still scarce. Our objective was to study the regulation of the NPY/melanocortin system in the adipose tissue (AT) and evaluate its therapeutic potential for obesity and type 2 diabetes. MethodsThe expression of the NPY/melanocortin receptors’ levels was assessed in the visceral AT of individuals with obesity and altered metabolism. Protein levels of these receptors were evaluated in cultured adipocytes incubated with ghrelin (30 and 100 ng/mL) and leptin (1 and 10 nM) and in the AT of an animal model with a mutation in the leptin receptor (ZSF1 rat), to understand their regulation by leptin and ghrelin. The vertical sleeve gastrectomy animal model was used to evaluate the putative therapeutic potential of the NPY/melanocortin system. ResultsIn this study, we unravelled that leptin (1 nM and 10 nM) selectively reduced the levels of NPY5R and MC3R but no other NPYR/MCRs in cultured adipocytes. In turn, acylated ghrelin (100 ng/mL) significantly increased NPY1R, but the inhibition of its receptor also abrogates MC3R levels. However, in the Lepr-deficient ZSF1 rat, both NPY5R and MC3R levels were reduced, along with other NPYRs and MCRs, suggesting that leptin resistance negatively affects NPY and melanocortin signalling. In human adipose tissue, we found a downregulation of genes encoding the NPY and melanocortin receptors in the visceral AT of individuals with obesity and insulin resistance, being correlated with genes regulating metabolic activity. Additionally, diabetic obese rats submitted to vertical sleeve gastrectomy showed increased levels of NPY, melanocortin, ghrelin, and leptin receptors in the AT, including MC3R, suggesting it may constitute a therapeutic target in obesity. ConclusionsOur results suggest that the AT NPY/melanocortin system, particularly the MC3R, may be involved in the neuroendocrine regulation of adipocyte metabolism. Altogether, our work shows MC3R is under the control of the ghrelin/leptin duo, is reduced in patients with obesity and prediabetes, and may constitute a therapeutic target in obesity.

Abstract B001: Macrophage adipocyte crosstalk as a node of intervention for pancreatic cancer-associated weight loss

Abstract Introduction: Up to 85% of patients with pancreatic ductal adenocarcinoma (PDAC) are afflicted with cachexia - a metabolic syndrome characterized by loss of skeletal muscle mass and adipose tissue wasting. Cachexia worsens patients’ quality of life, their ability to tolerate chemotherapy or surgical procedures, and, ultimately, their survival. Existing therapies often fail as the disease remains insufficiently understood. Drawing insights from obesity research, where changes in the local adipose immune environment play a crucial role in the pathophysiology, we explore the adipose tissue in the context of PDAC cachexia at single-cell resolution to uncover novel therapeutic avenues. Methods: We developed an ex vivo co-culture system to examine the effect of macrophages on adipocyte lipolysis by measuring the amount of released glycerol from adipocytes following incubation with macrophage supernatant. This robust platform can be used to interrogate factors directly affecting glycerol release from adipocytes. To identify candidate mediators of macrophage-adipocyte crosstalk, we profiled murine and human adipose tissue by flow cytometry and single-cell/nucleus RNA sequencing. The tissue samples were obtained from a cohort of genetically engineered KPC mice and a representative group of surgical patients. Patient adipose samples were collected intraoperatively without warm ischemic time at Heidelberg University Hospital. The cohort includes over 60 patients, divided into three groups: (1) PDAC with weight loss, (2) PDAC without weight loss, and (3) control patients without a history of malignancy. Of these, 25 samples are being used for single-nucleus RNA sequencing. The groups are balanced by sex and can be stratified into equally sized subgroups based on whether they had received neoadjuvant treatment. Results: Macrophage- conditioned media significantly elevated lipid release from murine adipocytes, demonstrating that macrophages may play a role in cancer-associated adipose wasting. Single-nucleus RNA sequencing data from our mice and from our human clinical cohort reveals distinct cell clusters with condition-specific subpopulations and differences unique to cancer-associated weight loss. The biggest phenotypic shifts were observed in macrophages and in adipocytes which showed signs of oxidative stress and altered metabolism associated with cachexia. Conclusion: We successfully established reliable workflows for processing and investigating murine and human AT in the context of PDAC cachexia. Our in vitro model highlights a distinct phenotype of murine macrophage supernatant inducing increased glycerol release from adipocytes. The in-depth analysis of our single-nucleus RNA sequencing data from our clinical cohort promises an unprecedentedly detailed and robust understanding of the human adipose immune environment, thereby enhancing our insights into PDAC cachexia. Citation Format:Felix P. Hambitzer, Farhad Faghihi, Brendan D. Parent, Michael Walsh, Susanne Roth, Andrew Aguirre, Christoph W. Michalski, Martin Loos, Stephanie K. Dougan, Evanna Mills, Max Heckler. Macrophage adipocyte crosstalk as a node of intervention for pancreatic cancer-associated weight loss [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B001.

Exploring herbal preconditioning strategies to improve adipose tissue stem cell therapy efficacy

The application of mesenchymal stem cells (MSCs), specifically those sourced from adipose tissue, has become a focal point in regenerative medicine, showing potential for effective cell therapy. Adipose-derived stem cells (ASCs) offer hope in treating various conditions, yet their viability and integration rates are hindered by the harsh post-transplantation environment marked by inflammation and oxidative stress at the injury site. Lawsonia inermis (henna), Zizyphus spina-christi (Christ's Thorn), and Glycyrrhiza glabra (licorice) are three plants known for their potent antioxidant properties primarily due to their rich content of phenolic compounds and flavonoids. Their potential health benefits make them valuable in both traditional medicine and modern therapeutic applications.This research delves into investigating and contrasting the effects of hydroalcoholic extracts from Lawsonia inermis, Zizyphus spina-christi, and Glycyrrhiza glabra on human adipose tissue stem cells as a pre-conditioning method to alleviate oxidative stress in cell therapy. ASCs were isolated from the pelvic cavity and abdomen, characterized using flow cytometry, and prompted to differentiate into adipogenic and osteogenic lineages. They were then subjected to different concentrations of the aforementioned herbal extracts at varying time frames, followed by MIC and MTT analysis. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and the Minimum Inhibitory Concentration (MIC) assay are both used to evaluate the effects of substances on cell viability and cell growth receptively. RNA extraction from ASCs and evaluation of antioxidant gene expression were conducted via Real-time PCR. Flow cytometry data confirmed the presence of MSC markers in the isolated cells from ASCs. Analysis of the herbal extract concentrations revealed that 50 ng/ml had a toxic effect on ASCs, establishing a toxicity reference point for adipose-derived MSCs. Real-time PCR results showcased a notable increase in the expression of antioxidant genes post-preconditioning with hydroalcoholic extracts. This study underscores, for the first time, the safety and lack of toxicity of preconditioning with these extracts for h.ASCs, enhancing their resilience and acclimatization under oxidative stress conditions, potentially boosting the therapeutic potential of h.ASCs. The antioxidant properties of Lawsonia inermis, Zizyphus spina-christi, and Glycyrrhiza glabra may enhance the expression of antioxidant genes (Sod, Gpx and Cata) through several mechanisms, including the activation of transcription factors like Nrf2 (Nuclear factor erythroid 2-related factor 2), which regulates antioxidant gene expression; modulation of signaling pathways such as MAPK (Mitogen-Activated Protein Kinase) and PI3K/Akt (Phosphoinositide 3-kinase/Protein Kinase B); and influencing epigenetic modifications like histone changes or DNA methylation that can alter gene expression. However, further research is necessary to clarify the specific mechanisms by which these plant extracts enhance antioxidant gene expression and to explore their potential therapeutic applications in combating oxidative stress-related diseases. Future proposals should consider more gene investigation and in vivo applications, as well as the long-term effects of pre-conditioning.

In vitro Chondrogenic Induction Promotes the Expression Level of IL-10 via the TGF-β/SMAD and Canonical Wnt/β-catenin Signaling Pathways in Exosomes Secreted by Human Adipose Tissue-derived Mesenchymal Stem Cells.

Current treatment approaches cannot exactly regenerate cartilage tissue. Regarding some problems encountered with cell therapy, exosomes are advantageous because of their "cell-free" nature. This study examines the relationship between IL-10 and TGF-β and Canonical Wnt/β-catenin signal pathways in human adipose tissue-derived MSCs exosomes (hAT-MSCs-Exos) after in vitro chondrogenic differentiation. Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and, as a control group, human fetal chondroblast cells (hfCCs) were differentiated chondrogenically in vitro. Exosome isolation and characterization analyses were performed. Chondrogenic differentiation was shown by Alcian Blue and Safranin O stainings. The expression levels of IL-10, TGF-β/SMAD signaling pathway genes, and Canonical Wnt/β-catenin signaling pathway genes, which play an essential role in chondrogenesis, were analyzed by RT-qPCR. Conditioned media cytokine levels were measured by using the TGF-β and IL-10 ELISA kits. IL-10 expression was upregulated in both chondrogenic differentiated hAT-MSC-Exos (dhAT-MSC-Exos) (p < 0.0001). In the TGF-β signaling pathway, TGF-β (p < 0.0001), SMAD2 (p < 0.0001), SMAD4 (p < 0.001), ACAN (p < 0.0001), SOX9 (p < 0.05) and COL1A2 (p < 0.0001) expressions were upregulated in dhAT-MSC-Exos. SMAD3 expression was upregulated in non-differentiated hAT-MSC-Exos. In the Canonical Wnt/β-catenin signaling pathway, WNT (p < 0.0001) and CTNNB1(p < 0.0001) expressions were upregulated in dhAT-MSC-Exos. AXIN (p < 0.0001) expression was upregulated in non-differentiated hAT-MSC-Exos. TGF-β and IL-10 levels were higher in dhAT-MSCs) (p < 0.0001). Related to these results, IL-10 may induce TGF-β/SMAD and Canonical Wnt/β-catenin signaling pathways in hAT-MSC exosomes obtained after chondrogenic differentiation. Therefore, using these exosomes for cartilage regeneration can lead to the development of treatment methods.

Sox2-mediated transdifferentiation of hAT-MSCs into induced neural progenitor-like cells for remyelination therapies

Mesenchymal stem cells (MSCs) are converted to neural cells using growth factors and chemicals. Although these neural cells are effective at modulating disease symptoms, they are less effective at replacing lost neural cells. Direct transdifferentiation seems to be a promising method for generating the required cells for regenerative medicine applications. Sox2 is a key transcription factor in neural progenitor (NP) fate determination and has been frequently used for transdifferentiating different cell types to NPs. Here, we demonstrated that the overexpression of a single transcription factor, Sox2, in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) led to the generation of induced NPs-like cells that were clonogenic, proliferative and passageable, and showed the potential to differentiate into three neural lineages. NPs are known as progenitors with the potential to differentiate into oligodendrocytes. In vivo, following transplantation into demyelinated adult mouse brains, they survived, differentiated and integrated into the adult brain while participating in the remyelination process and behavioral improvement. This report introduces a beneficial, low-cost and effective approach for generating NPs from an accessible adult source for autologous applications in treating neurodegenerative diseases, including remyelination therapies for multiple sclerosis and other demyelinating diseases.