ABSTRACT: we analyzed the expression level change of transcription factors in adipose derived stem cells during osteogenic differentiation and found a candidate target gene, Sox11. We defined that Sox11 suppresses osteogenic differentiation through overexpression and knock down of Sox11. total RNA obtained from adipose derived stem cells subjected to 1,3,6,10 or 14 days in osteogenic differentiation compared to undifferentiated control adipose derived stem cells.
Project description:we analyzed the expression level change of transcription factors in adipose derived stem cells during osteogenic differentiation and found a candidate target gene, Sox11. We defined that Sox11 suppresses osteogenic differentiation through overexpression and knock down of Sox11.
Project description:Adipose tissue harbours a significant number of multipotent adult stem cells of mesenchymal origin known as adipose-derived stem cells (ADSCs). Broad differentiation potential and convenient accessibility of ADSCs make them an attractive source of adult mesenchymal stem cell for regenerative medicine and cell developmental plasticity research. Genome-wide microarray expression profiling was performed to identify genes deregulated during osteogenic differentiation of ADSCs to evaluate developmental plasticity of these cells. Dynamics of epigenetic modifications were analyzed in parallel and associated with the gene expression profile. Gene expression profile was analyzed in adipose-derived stem cells (ADSCs) differentiated into osteogenic lineage from 3 donors and compared to undifferentiated cells from the same donors.
Project description:Ability to perform osteogenic differentiation is one of the minimal criteria of mesenchymal stem cells (MSCs). Still, it is generally unknown whether osteogenic differentiation is universal cell fate or various phenotypically similar cell states. Besides this, MSCs and their secretomes are actively using for cell/cell-free therapy development, but systemic inter-source variation in MSCs secretomes, proteomes and differentiation mechanisms are still poorly understood. Therefore, here we compared proteomic and secretomic profiles of human mesenchymal cells from six sources: osteoblasts (bone), WJ-MSCs (Warton’s jelly), AD-MSCs (adipose), PDLSCs (tooth: Periodontal Ligament Stem Cells), DPSCs (tooth: Dental Pulp Stem Cells) and GFs (tooth: Gingival Fibroblasts). For experiments we used cells in early passages (3-5) isolated from 3-6 individuals. All cells were compared in standard cultivation and in the 10th day after induction of osteogenic differentiation.
Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. To analyse whether the BM-MSC and Fat-MSC (ASC) differentiation stage may define the sarcoma phenotype, RbloxP/loxPp53loxP/loxP BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage and both Rb and p53 were excised with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre) at different stages (day 0 and 10) along osteogenic differentiation. NSG mice were inoculated subcutaneously with 5M-CM-^W10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using: WT BM-MSCs and ASCs, Rb-/-p53-/- BM-MSCs and ASCs previously differentiated to the osteogenic lineage for 10 days and a tumor cell line derived from p53-/-Rb-/- BM-MSC differentiated to the osteogenic lineage for 10 days.
Project description:Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases, including osteoporosis and bone tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (MSCs). CASC2 knockdown using small interfering RNA (siCASC2) increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining levels, or the expression of early osteogenic transcripts including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at either transcriptional and protein level. Inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP levels. The results indicate that lncRNA CASC2 regulates late osteogenesis and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests lncRNA CASC2 as a potential new therapeutic target in bone mineralization.
Project description:The aim of this study is to characterize how the extracellular matrix secreted by adipose-derived stem cells (ADSC) during osteogenesis affects the differentiation process. Specifically, ADSC undergo osteogenesis by following similar maturational phases as bone marrow-derived stem cells. However, it is unclear how the differentiation process is the same and how it differs. We first focused on ADSC behavior by analyzing whole transcriptome changes in response to osteogenic media supplements added into the tissue culture medium. We then developed osteogenic differentiation expression profiles for the ADSC and identify key genes and pathways that serve as an osteogenesis signature. This expression signature acted as a template for comparison of how extracellular matrix (ECM) affected ADSC differentiation. We studied ADSC induced to differentiate on ECM isolated from day 16 in the differentiation process (the midpoint in osteogenesis) as well as ECM from day 11 in the differentiation process. Ultimately, we aim to dissect the relationship between cells grown on ECM as an in vitro growth substrate and cells grown on tissue culture plastic; both in the presence of osteogenic supplements. This study, will allow us to determine the extent to which ECM affects the differentiation process.
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:Vascular calcification is common pathology various forms of which presented in the half of humans. Calcific aortic valve disease (CAVD) is one of the most dangerous forms of vascular calcification. Despite high mortality there is no target therapy against CAVD. Thus, we tested crenigacestat (LY3039478), inhibitor of Notch-signaling, and shRNA against RBPJk for inhibition of osteogenic differentiation of velve intersticial cells (VICs) in vitro. Both of them effectivelly enhibited osteogenic differentiation and we performed proteomics analysis do describe molecular mechanisms of their effect. Presented dataset contain results of shotgun proteomics analysis with ion mobility in TimsToF Pro instrument (in DDA PASEF mode) of VICs in classic osteogenic medium (OM) and OM supplemented with crenigacestat, DMSO or shCSL.
Project description:Cartilage endplate-derived stem cells (CESCs) with chondro-osteogenic differentiation capacity may be responsible for the balance of chondrification and ossification in cartilage endplate (CEP). CEP is an avascular and hypoxic tissue, and hypoxia could inhibit the osteogenic differentiation of CESCs. We used high-throughput scanning to identify differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) during osteogenic differentiation of CESCs under hypoxia compared to those induced under normoxia. Human cartilage endplate-derived stem cells (CESCs) were treated with osteogenic differentiation medium under normoxia and hypoxia for 21 days respectively.
Project description:The purpose of this study was to examine the effects of hDPCs-CM on osteogenic differentiation of hDFCs and to compare gene expression in hDFCs in the presence or absence of hDPCs-CM Total RNAs were isolated from hDFCs in osteogenic medium for 7 days with or without hDPCs-CM.