Comparison of stromal vascular fraction cell composition between Coleman fat and extracellular matrix/stromal vascular fraction gel.

As a mechanically condensed product of Coleman fat, extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) eliminates adipocytes, concentrates SVF cells, and improves fat graft retention. This study aims to compare SVF cell composition between Coleman fat and ECM/SVF-gel. Matched Coleman fat and ECM/SVF-gel of 28 healthy women were subjected to RNA-seq, followed by functional enrichment and cell-type-specific enrichment analyses, and deconvolution of SVF cell subsets, reconstructing SVF cell composition in the transcriptome level. ECM/SVF-gels had 9 upregulated and 73 downregulated differentially expressed genes (DEGs). Downregulated DEGs were mainly associated with inflammatory and immune responses, and enriched in fat macrophages. M2 macrophages, resting CD4+ memory T cells, M1 macrophages, resting mast cells, and M0 macrophages ranked in the top five most prevalent immune cells in the two groups. The proportions of the principal non-immune cells (e.g., adipose-derived stem cells, pericytes, preadipocytes, microvascular endothelial cells) had no statistical differences between the two groups. Our findings reveal ECM/SVF-gels share the same dominant immune cells beneficial to fat graft survival with Coleman fat, but exhibiting obvious losses of immune cells (especially macrophages), while non-immune cells necessary for adipose regeneration might have no significant loss in ECM/SVF-gels and their biological effects could be markedly enhanced by the ECM/SVF-gel's condensed nature.

Characterization and functional analysis of the adipose tissue-derived stromal vascular fraction of pediatric patients with osteogenesis imperfecta.

Pediatric patients with Osteogenesis Imperfecta (OI), a heritable connective tissue disorder, frequently suffer from long bone deformations. Surgical correction often results in bone non-unions, necessitating revision surgery with autogenous bone grafting using bone-marrow-derived stem cells (BM-SC) to regenerate bone. BM-SC harvest is generally invasive and limited in supply; thus, adipose tissue's stromal vascular fraction (SVF) has been introduced as an alternative stem cell reservoir. To elucidate if OI patients' surgical site dissected adipose tissue could be used as autologous bone graft in future, we investigated whether the underlying genetic condition alters SVF's cell populations and in vitro differentiation capacity. After optimizing SVF isolation, we demonstrate successful isolation of SVF of pediatric OI patients and non-OI controls. The number of viable cells was comparable between OI and controls, with about 450,000 per gram tissue. Age, sex, type of OI, disease-causing collagen mutation, or anatomical site of harvest did not affect cell outcome. Further, SVF-containing cell populations were similar between OI and controls, and all isolated SVF's demonstrated chondrogenic, adipogenic, and osteogenic differentiation capacity in vitro. These results indicate that SVF from pediatric OI patients could be used as a source of stem cells for autologous stem cell therapy in OI.

Adipocyte Heterogeneity Underlying Adipose Tissue Functions.

Adipose tissue distribution in the human body is highly heterogeneous, and the relative mass of different depots is differentially associated with metabolic disease risk. Distinct functions of adipose depots are mediated by their content of specialized adipocyte subtypes, best exemplified by thermogenic adipocytes found in specific depots. Single-cell transcriptome profiling has been used to define the cellular composition of many tissues and organs, but the large size, buoyancy, and fragility of adipocytes have rendered it challenging to apply these techniques to understand the full complexity of adipocyte subtypes in different depots. Discussed here are strategies that have been recently developed for investigating adipocyte heterogeneity, including single-cell RNA-sequencing profiling of the stromal vascular fraction to identify diverse adipocyte progenitors, and single-nuclei profiling to characterize mature adipocytes. These efforts are yielding a more complete characterization of adipocyte subtypes in different depots, insights into the mechanisms of their development, and perturbations associated with different physiological states such as obesity. A better understanding of the adipocyte subtypes that compose different depots will help explain metabolic disease phenotypes associated with adipose tissue distribution and suggest new strategies for improving metabolic health.

PEX13 is required for thermogenesis of white adipose tissue in cold-exposed mice.

Non-shivering thermogenesis (NST) is a heat generating process controlled by the mitochondria of brown adipose tissue (BAT). In the recent decade, 'functionally' acting brown adipocytes in white adipose tissue (WAT) has been identified as well: the so-called process of the 'browning' of WAT. While the importance of uncoupling protein 1 (UCP1)-oriented mitochondrial activation has been intensely studied, the role of peroxisomes during the browning of white adipocytes is poorly understood. Here, we assess the change in peroxisomal membrane proteins, or peroxins (PEXs), during cold stimulation and importantly, the role of PEX13 in the cold-induced remodeling of white adipocytes. PEX13, a protein that originally functions as a docking factor and is involved in protein import into peroxisome matrix, was highly increased during cold-induced recruitment of beige adipocytes within the inguinal WAT of C57BL/6 mice. Moreover, beige-induced 3 T3-L1 adipocytes and stromal vascular fraction (SVF) cells by exposure to the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone showed a significant increase in mitochondrial thermogenic factors along with peroxisomal proteins including PEX13, and these were confirmed in SVF cells with the beta 3 adrenergic receptor (ß3AR)-selective agonist CL316,243. To verify the relevance of PEX13, we used the RNA silencing method targeting the Pex13 gene and evaluated the subsequent beige development in SVF cells. Interestingly, siPex13 treatment suppressed expression of thermogenic proteins such as UCP1 and PPARγ coactivator 1 alpha (PGC1α). Overall, our data provide evidence supporting the role of peroxisomal proteins, in particular PEX13, during beige remodeling of white adipocytes.

Expression of the Adipocyte Progenitor Markers MSCA1 and CD36 is Associated With Adipose Tissue Function in Children.

CONTEXT: MSCA1 (mesenchymal stem cell antigen 1) and CD36 (cluster of differentiation 36) have been described as novel adipocyte progenitor markers in adults with a potential relevance for obesity and adipocyte progenitor function. OBJECTIVE: With the early manifestation of obesity in children and formation of adipose tissue (AT) dysfunction, children provide the opportunity to characterize the function of MSCA1 and CD36 during physiological AT accumulation and with obesity and related disease. METHODS: We investigated MSCA1 and CD36 expression in adipocytes and stroma vascular fraction (SVF) cells from 133 children of the Leipzig AT Childhood cohort with regard to AT accumulation and biology. In a subsample we analyzed how MSCA1 and CD36 expression is related to adipose progenitor capacities in vitro (ie, proliferation, differentiation and mitochondrial function). RESULTS: Both MSCA1 and CD36 are differentially expressed in adipocytes and SVF cells of children. MSCA1 expression is positively correlated to obesity-associated AT dysfunction (ie, adipocyte hypertrophy and serum high-sensitivity C-reactive protein), and high SVF MSCA1 expression is associated with increased mitochondrial respiration in vitro. CD36 expression is not associated with AT dysfunction but SVF CD36 expression is downregulated in children with overweight and obesity and shows a positive association with the differentiation capacity of SVF cells ex vivo and in vitro. CONCLUSION: Both MSCA1 and CD36 are associated with obesity-related alterations in AT of children. In particular, CD36 expression predicts adipogenic potential of SVF cells, indicating a potential role in the regulation of adipocyte hyperplasia and hypertrophy with obesity development in children.

Polychromatic Flow Cytometric Analysis of Stromal Vascular Fraction from Lipoaspirate and Microfragmented Counterparts Reveals Sex-Related Immunophenotype Differences.

Mesenchymal stem/stromal cells or medicinal signaling cells (MSC)-based therapy holds promise as a beneficial strategy for treating knee OA (osteoarthritis), but there is no standardized protocols nor mechanistic understanding. In order to gain a better insight into the human MSC from adipose tissue applied for autologous OA treatment, we performed extensive comparative immunophenotyping of the stromal vascular fraction from lipoaspirate or microfragmented lipoaspirates by polychromatic flow cytometry and investigated the cellular components considered responsible for cartilage regeneration. We found an enrichment of the regenerative cellular niche of the clinically applied microfragmented stromal vascular fraction. Sex-related differences were observed in the MSC marker expression and the ratio of the progenitor cells from fresh lipoaspirate, which, in female patients, contained a higher expression of CD90 on the three progenitor cell types including pericytes, a higher expression of CD105 and CD146 on CD31highCD34high endothelial progenitors as well as of CD73 on supra-adventitialadipose stromal cells. Some of these MSC-expression differences were present after microfragmentation and indicated a differential phenotype pattern of the applied MSC mixture in female and male patients. Our results provide a better insight into the heterogeneity of the adipose MSC subpopulations serving as OA therapeutics, with an emphasis on interesting differences between women and men.

Culturing patient-derived malignant hematopoietic stem cells in engineered and fully humanized 3D niches.

Human malignant hematopoietic stem and progenitor cells (HSPCs) reside in bone marrow (BM) niches, which remain challenging to explore due to limited in vivo accessibility and constraints with humanized animal models. Several in vitro systems have been established to culture patient-derived HSPCs in specific microenvironments, but they do not fully recapitulate the complex features of native bone marrow. Our group previously reported that human osteoblastic BM niches (O-N), engineered by culturing mesenchymal stromal cells within three-dimensional (3D) porous scaffolds under perfusion flow in a bioreactor system, are capable of maintaining, expanding, and functionally regulating healthy human cord blood-derived HSPCs. Here, we first demonstrate that this 3D O-N can sustain malignant CD34+ cells from acute myeloid leukemia (AML) and myeloproliferative neoplasm patients for up to 3 wk. Human malignant cells distributed in the bioreactor system mimicking the spatial distribution found in native BM tissue, where most HSPCs remain linked to the niches and mature cells are released to the circulation. Using human adipose tissue-derived stromal vascular fraction cells, we then generated a stromal-vascular niche and demonstrated that O-N and stromal-vascular niche differentially regulate leukemic UCSD-AML1 cell expansion, immunophenotype, and response to chemotherapy. The developed system offers a unique platform to investigate human leukemogenesis and response to drugs in customized environments, mimicking defined features of native hematopoietic niches and compatible with the establishment of personalized settings.

The capacity of differentiation of stromal vascular fraction cells into beige adipocytes is markedly reduced in subjects with overweight/obesity and insulin resistance: effect of genistein.

BACKGROUND: Dietary bioactive compounds have been demonstrated to produce several health benefits. Genistein, an isoflavone of soy protein, and resveratrol, a polyphenol from grapes, have been shown to improve insulin sensitivity and to stimulate white adipose tissue (WAT) browning, leading to increased energy expenditure. However, it has not been demonstrated in humans whether genistein or resveratrol have the capacity to stimulate the differentiation of stromal vascular fraction (SVF) cells from white fat into beige adipocytes. SUBJECTS/METHODS: With this aim, we assessed whether stromal vascular fraction cells obtained from biopsies of the subdermal fat depots of subjects with normal body weight (NW) or from subjects with overweight/obesity with (OIR) or without (OIS) insulin resistance were able to differentiate into the beige adipose tissue lineage in vitro, by exposing the cells to genistein, resveratrol, or the combination of both. RESULTS: The results showed that SVF cells obtained from NW or OIS subjects were able to differentiate into beige adipocytes according to an increased expression of beige biomarkers including UCP1, PDRM-16, PGC1α, CIDEA, and SHOX2 upon exposure to genistein. However, SVF cells from OIR subjects were unable to differentiate into beige adipocytes with any of the inducers. Exposure to resveratrol or the combination of resveratrol/genistein did not significantly stimulate the expression of browning markers in any of the groups studied. We found that the non-responsiveness of the SVF from subjects with obesity and insulin resistance to any of the inducers was associated with an increase in the expression of endoplasmic reticulum stress markers. CONCLUSION: Consumption of genistein may stimulate WAT browning mainly in NW or OIS subjects. Thus, obesity associated with insulin resistance may be considered as a condition that prevents some beneficial effects of some dietary bioactive compounds.