Project description:Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways. Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm2), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm2) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis. Therefore, we conclude that cytoskeletal tension is important for CTGF-regulated ASC osteogenic differentiation. Computed
Project description:Circulating osteoprogenitor (COP) are a population of cell in the peripheral circulation that possess functional and phenotypical characteristics of multipotent stromal cells (MSCs). While there is functional overlap, it is not known how COP cells are related to bone marrow (BM)-derived MSCs (BM-MSCs) and other better characterized stromal progenitor populations such as adipose-derived stromal cells (ASCs). This study compares COP cells to BM-MSCs and ASCs through detailed transcriptomic and proteomic analyses. COP cells have a distinct gene and protein expression pattern to BM-MSCs and ASCs, with a significantly stronger immune footprint, likely owing to their hematopoietic lineage. However, they also have a similar pattern of expression BM-MSCs and ASCs, in genes and proteins in progenitor cell differentiation and proliferation pathways. This study shows COP cells to be a unique but functionally similar population to BM-MSCs and ASCs, sharing their proliferation and differentiation capacity, but with a strong immune phenotype, with potential for translational regenerative medicine strategies.
Project description:Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype by microarray analysis.
Project description:Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways. Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm2), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm2) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis. Therefore, we conclude that cytoskeletal tension is important for CTGF-regulated ASC osteogenic differentiation.
Project description:We used the resolving power of single-cell transcriptional profiling to molecularly characterize the mouse adipose stem and progenitor cell-enriched, subcutaneous adipose stromal vascular fraction. We molecularly assessed CD45- CD31- SVF cells using the 10x Genomics Chromium (10x) platform.
Project description:Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype by microarray analysis. Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7d: early stage) and 21 days (21d: late stage) of adipocyte differentiation in vitro.
Project description:The treatment of bone defects caused by infection, trauma or neoplasms remains a clinical challenge. Autologous bone transplantation is limited by availability, donor site morbidity and surgical risk factors. This has given rise to stromal/stem-cell based therapy. Bone marrow derived stromal cells (BMSCs) have been studied to a large extent and show high regenerative potential but their use is limited by availability, donor site morbidity and the relatively low cell yield as they represent only <0.1% of cell harvested from bone marrow aspirate. At the same time, they are the closest mesenchymal stromal cells for bone tissue engineering given their tissue origin and, unlike other mesenchymal stromal cells, can support the formation of hematopoietic marrow. Adipose tissue derived stromal cells (ASCs) as part of the stromal vascular fraction of adipose tissue can as well undergo osteogenic differentiation but can be additionally isolated in a sufficient quantity from lipoaspirate after liposuction of abundant subcutaneous fat tissue. Here, it has been shown that there are no major differences in regard to proliferation or differentiation capacity of ASCs derived from subcutaneous fat of different anatomical regions. It has been shown that BMSCs are more prone to senescence during expansion and passage than ASCs and that ageing impacts proliferative capabilities of BMSCs more than that of ASCs while it has also been reported that osteogenic differentiation capacity is least impacted by age. Multiple studies have compared the characteristics of these two mesenchymal stromal cells in regard to bone tissue engineering in vitro. Most studies point to inferior extracellular matrix mineralization and lower expression of key osteogenic transcription markers like Runx2 in osteogenic differentiated ASCs compared to BMSCs. On the other hand, a study by Rath et al. found contrary results using particular culturing conditions like 3D bioglass scaffolds. An intraindividual comparison of human MSCs of three donors cultured on decellularized porcine bone confirmed superior osteogenic capacity of BMSCs compared to ASCs. In contrast to BMSCs, ASCs were not able to induce heterogenic ossification in a mouse model. In a sheep tibia defect model application of BMSCs resulted in a significantly higher amount of newly formed bone tissue. Importantly, Osteogenic differentiated ASCs do not support the formation of a hematopoietic marrow. Proteomics enables large-scale analysis of proteins present in a cell type and can be used to identify differentially regulated key proteins in a comparative approach. A comparative proteomic analysis of BMSCs and ASCs by Roche et al. in 2009 identified 556 proteins with 78% of these not being differentially regulated between these two cell populations, regarded as high similarity. Another comparative proteomic study of 2016 by Jeon et al. found 90 differentially regulated proteins out of 3000 total identified proteins. Both studies do not specify a number of different tissue donors and in part using cell lines. Looking for differences upon osteogenic differentiation, transcriptomic comparison of osteogenic differentiated porcine ASCs and BMSCs has been performed, resulting in 21 differentially expressed genes after 21 days of osteogenic culture conditions. Still, it remains unanswered, which are the key distinctive features of osteogenic differentiated ASCs and BMSCs at protein level that might help address the abovementioned weaknesses of ASCs in bone tissue engineering/regeneration for translational research. To overcome this need, an intraindividual comparative DIA based proteomic analysis of osteogenic differentiated human BMSC and ASCs was performed in this study.
Project description:Adipose-derived stromal/stem cells (ASC) capable of multipotential differentiation can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our recent experience isolating and immunophenotypically characterizing ASCs from >60 human subjects
Project description:Pathological expansion of adipose tissue (AT) in obesity is supported by adipocyte precursors, termed adipose-derived stromal/stem cells (ASCs). Elucidation of mechanisms underlying ASC function may lead to therapeutic interventions to treat fat mass accumulation. Using epigenome-wide association studies, we explored the impact of obesity on the methylation signature of human ASCs.