Single-Cell Transcriptome Analysis of Human Adipose-Derived Stromal Cells Identifies a Contractile Cell Subpopulation.
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ABSTRACT:
SUBMITTER: Wolmarans E
PROVIDER: S-EPMC8102097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mesenchymal stem/stromal cells (MSCs) exhibit multidifferentiation potential, paralleled with immunomodulatory and trophic properties that make them viable alternative tools for the treatment of degenerative disorders, allograft rejection, autoimmune diseases, and tissue regeneration. MSC functional attributes can be modulated by exposing them to inflammatory-stimulating microenvironments (i.e., priming) before their therapeutic use. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that plays key roles in immune response and hematopoiesis modulation through direct effects on hematopoietic progenitors' proliferation, survival, and mobilization. Despite the established roles of MSCs supporting hematopoiesis, the effects of G-CSF on MSCs biology have not been thoroughly explored. This study reveals that G-CSF has also direct effects on adipose-derived MSCs (ADSCs), modulating their functions. Herein, microarray-based transcriptomic analysis shows that G-CSF stimulation in vitro results in modulation of various signaling pathways including ones related with the metabolism of hyaluronan (HA), conferring a profile of cell mobilization to ADSCs, mediated in a cell-intrinsic fashion in part by reducing CD44 expression and HA synthesis-related genes. Collectively, these data suggest a direct modulatory effect of G-CSF on ADSC function, potentially altering their therapeutic capacity and thus the design of future clinical protocols.
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Project description:Cardiac tissue engineering strategies have the potential to regenerate functional myocardium following myocardial infarction. In this study, we utilized novel electrospun fibrin microfiber sheets of different stiffnesses (50.0?±?11.2 kPa and 90.0?±?16.4 kPa) to engineer biomimetic models of vascularized cardiac tissues. We characterized tissue assembly, electrophysiology, and contractility of neonatal rat ventricular cardiomyocytes (NRVCMs) cultured on these sheets. NRVCMs cultured on the softer substrates displayed higher conduction velocities (CVs) and improved electrophysiological properties. Human umbilical vein endothelial cells (HUVECs) formed dense networks on the sheets when co-cultured with human adipose-derived stem/stromal cells (hASCs). To achieve vascularized cardiac tissues, we tested various tri-culture protocols of NRVCM:hASC:HUVEC and found that a ratio of 1,500,000:37,500:150,000 cells/cm2 enabled the formation of robust endothelial networks while retaining statistically identical electrophysiological characteristics to NRVCM-only cultures. Tri-cultures at this ratio on 90 kPa substrates exhibited average CVs of 14?±?0.6?cm/s, Action Potential Duration (APD)80 and APD30 of 152?±?11?ms and 71?±?6?ms, respectively, and maximum capture rate (MCR) of 3.9?±?0.7?Hz. These data indicate the significant potential of generating densely packed endothelial networks together with electrically integrated cardiac cells in vitro as a physiologic 3D cardiac model.
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Project description:Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for articular cartilage repair and regeneration. However, the terminal hypertrophic differentiation of Ad-MSC-derived cartilage is a critical barrier during hyaline cartilage regeneration. In this study, we investigated the role of matrilin-3 in preventing Ad-MSC-derived chondrocyte hypertrophy in vitro and in an osteoarthritis (OA) destabilization of the medial meniscus (DMM) model. Methacrylated hyaluron (MAHA) (1%) was used to encapsulate and make scaffolds containing Ad-MSCs and matrilin-3. Subsequently, the encapsulated cells in the scaffolds were differentiated in chondrogenic medium (TGF-?, 1-14 days) and thyroid hormone hypertrophic medium (T3, 15-28 days). The presence of matrilin-3 with Ad-MSCs in the MAHA scaffold significantly increased the chondrogenic marker and decreased the hypertrophy marker mRNA and protein expression. Furthermore, matrilin-3 significantly modified the expression of TGF-?2, BMP-2, and BMP-4. Next, we prepared the OA model and transplanted Ad-MSCs primed with matrilin-3, either as a single-cell suspension or in spheroid form. Safranin-O staining and the OA score suggested that the regenerated cartilage morphology in the matrilin-3-primed Ad-MSC spheroids was similar to the positive control. Furthermore, matrilin-3-primed Ad-MSC spheroids prevented subchondral bone sclerosis in the mouse model. Here, we show that matrilin-3 plays a major role in modulating Ad-MSCs' therapeutic effect on cartilage regeneration and hypertrophy suppression.
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Project description:BACKGROUND:As the population ages, an increasing number of postmenopausal women are donors of adipose stromal cells (ASCs) and may benefit from autologous ASC-related treatments. However, the effect of menopausal status on ASCs has not been investigated. METHODS:RNA sequencing data were downloaded, and differentially expressed genes (DEGs) were identified. Hierarchical clustering, Gene Ontology, and pathway analyses were applied to the DEGs. Two gene coexpression network analysis approaches were applied to the DEGs to provide a holistic view and preserve gene interactions. Hub genes of the gene coexpression network were identified, and their expression profiles were examined with clinical samples. ASCs from pre- and postmenopausal women were co-cultured with monocytes and T cells to determine their immunoregulatory role. RESULTS:In total, 2299 DEGs were identified and presented distinct expression profiles between pre- and postmenopausal women. Gene Ontology and pathway analyses revealed some fertility-, sex hormone-, immune-, aging-, and angiogenesis-related terms and pathways. Gene coexpression networks were constructed, and the top hub genes, including TIE1, ANGPT2, RNASE1, PLVAP, CA2, and MPZL2, were consistent between the two approaches. Expression profiles of hub genes from the RNA sequencing data and clinical samples were consistent. ASCs from postmenopausal women elicit M1 polarization, while their counterparts facilitate CD3/4+ T cell proliferation. CONCLUSIONS:The present study reveals the transcriptome differences in ASCs derived from pre- and postmenopausal women and provides holistic views by preserving gene interactions via gene coexpression network analysis. The top hub genes identified by this study could serve as potential targets to enhance the therapeutic potential of ASCs.
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Project description:A central function of adipose tissue is in the management of systemic energy homeostasis that is achieved through the co-ordinated regulation of energy storage and mobilization, adipokine release, and immune functions. With the dramatic increase in the prevalence of obesity and obesity-related metabolic disease over the past 30 years, there has been extensive interest in targeting adipose tissue for therapeutic benefit. However, in order for this goal to be achieved it is essential to establish a comprehensive atlas of adipose tissue cellular composition and define mechanisms of intercellular communication that mediate pathologic and therapeutic responses. While traditional methods, such as fluorescence-activated cell sorting (FACS) and genetic lineage tracing, have greatly advanced the field, these approaches are inherently limited by the choice of markers and the ability to comprehensively identify and characterize dynamic interactions among stromal cells within the tissue microenvironment. Single cell RNA sequencing (scRNAseq) has emerged as a powerful tool for deconvolving cellular heterogeneity and holds promise for understanding the development and plasticity of adipose tissue under normal and pathological conditions. scRNAseq has recently been used to characterize adipose stem cell (ASC) populations and has provided new insights into subpopulations of macrophages that arise during anabolic and catabolic remodeling in white adipose tissue. The current review summarizes recent findings that use this technology to explore adipose tissue heterogeneity and plasticity.
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Project description:The complex relationship between metabolic disease risk and body fat distribution in humans involves cellular characteristics which are specific to body fat compartments. Here we show depot-specific differences in the stromal vascual fraction of visceral and subcutaneous adipose tissue by performing single-cell RNA sequencing of tissue specimen from obese individuals. We characterize multiple immune cells, endothelial cells, fibroblasts, adipose and hematopoietic stem cell progenitors. Subpopulations of adipose-resident immune cells are metabolically active and associated with metabolic disease status and those include a population of potential dysfunctional CD8+ T cells expressing metallothioneins. We identify multiple types of adipocyte progenitors that are common across depots, including a subtype enriched in individuals with type 2 diabetes. Depot-specific analysis reveals a class of adipocyte progenitors unique to visceral adipose tissue, which shares common features with beige preadipocytes. Our human single-cell transcriptome atlas across fat depots provides a resource to dissect functional genomics of metabolic disease.
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Project description:BACKGROUND:Studies have demonstrated that human adipose-derived stromal cells (ASCs) are able to repair acute calvarial injuries. The more clinically relevant repair of an established skeletal defect, however, has not been addressed. The authors sought to determine whether human ASCs could heal chronic (established) calvarial defects. METHODS:Critical-sized (4 mm) mouse parietal defects were created. Human ASCs were engrafted either immediately postoperatively (acute defect) or 8 weeks following defect creation (established defect). Methods of analysis included microcomputer tomography scans, histology, and in situ hybridization. Finally, human ASCs were treated in vitro with platelet-rich plasma to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (alkaline phosphatase, alizarin red, and quantitative reverse transcriptase polymerase chain reaction). RESULTS:Nearly complete osseous healing was observed when calvarial defects were immediately engrafted with human ASCs. In contrast, when human ASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microcomputer tomography, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in an established human ASC-engrafted defect. In situ hybridization and quantitative reverse transcriptase polymerase chain reaction showed an increase in bone morphogenetic protein (BMP) expression (BMP-2, BMP-4, and BMP-7) acutely following calvarial defect creation. Finally, in vitro treatment of human ASCs with platelet-rich plasma enhanced osteogenic differentiation and increased BMP-2 expression. CONCLUSIONS:Although human ASCs can be utilized to heal an acute mouse calvarial defect, they do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated after injury may explain these differences in healing. Platelet-rich plasma enhances osteogenic differentiation of human ASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.
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Project description:The application of appropriate cell origin for utilizing in regenerative medicine is the major issue. Various kinds of stem cells have been used for the tissue engineering and regenerative medicine. Such as, several stromal cells have been employed as treat option for regenerative medicine. For example, human bone marrow-derived stromal cells and adipose-derived stromal cells (ADSCs) are used in cell-based therapy. Data relating to the stem cell therapy and processes associated with ADSC has developed remarkably in the past 10 years. As medical options, both the stromal vascular and ADSC suggests good opportunity as marvelous cell-based therapeutics. The some biological features are the main factors that impact the regenerative activity of ADSCs, including the modulation of the cellular immune system properties and secretion of bioactive proteins such as cytokines, chemokines and growth factors, as well as their intrinsic anti-ulcer and anti-inflammatory potential. A variety of diseases have been treated by ADSCs, and it is not surprising that there has been great interest in the possibility that ADSCs might be used as therapeutic strategy to improve a wider range of diseases. This is especially important when it is remembered that routine therapeutic methods are not completely effective in treat of diseases. Here, it was discuss about applications of ADSC to colitis, liver failure, diabetes mellitus, multiple sclerosis, orthopaedic disorders, hair loss, fertility problems, and salivary gland damage.
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Project description:Fat grafting can reduce radiation-induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose-derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74- and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor β3 (TGF-β3) and decreased levels of TGF-β1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74- or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation-induced fibrosis.
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Project description:Introduction:Many pathological conditions may benefit from cell therapy using mesenchymal stromal cells, particularly from adipose tissue (ASCs). Cells may be grafted in an environment with a remnant polymicrobial component. The aim is to investigate the behavior of ASCs when brought in contact with a large panel of bacteria. Materials and Methods:Carboxyfluorescein-labelled bacterial interaction with ASCs was followed by confocal time-lapse microscopy. Costaining with LAMP-1 was also analyzed. Viability of 4 gram-negative and 4 gram-positive bacterial strains after 6?h of coculture with ASCs was assessed by agar colony counting and by flow cytometry using SYTO-62®/propidium iodide (PI) for membrane permeabilization and DiOC6 for depolarization. A murine model of periodontitis was used to assess in vivo antibacterial capacities of ASCs. Results:A significant increase of PI-positive events for all bacterial strains and an increase of the DiOC6 signal were obtained after contact with ASCs. The number of CFU was also significantly decreased for several bacterial strains. 0.4 ?m transwell systems illustrated the necessary direct contact to induce maximal bacterial membrane damages. Some bacteria were observed into phagolysosomes, confirming macrophage-like properties of ASCs. In vivo, the bacterial load was significantly lower in the ASC-grafted side compared to the control. Conclusion:Our results highlight for the first time a broad range of antibacterial actions of ASCs, by phagocytosis, secretion of oxygenated free radicals and antibacterial molecules. These data are in line with the development of new therapeutic strategies based on ASC transplantation, appropriated in immune-dysbiotic tissue context such as periodontitis or chronic wounds.
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