Simvastatin enhances proliferation and pluripotent gene expression by canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) in vitro.
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ABSTRACT:
SUBMITTER: Nantavisai S
PROVIDER: S-EPMC6820287 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundReported efficacy of platelet-rich plasma (PRP) in regenerative medicine is contradictory. We validated the effects of PRP on proliferation of canine bone marrow-derived multipotent mesenchymal stromal cells (K9BMMSCs) in vitro. PRP was extracted from blood of six dogs with osteoarthritis. K9BMMSCs were established from bone marrow and characterized for CD90 and CD19 expression by immunocytochemistry. Effects of PRP concentrations on viability of matching autologous K9BMMSCs were validated using MTS assay.ResultsPositive CD90 and negative CD19 expression confirmed MSC origin. PRP at 40% volume/volume concentration increased, while PRP at 80 and 100% v/v concentrations suppressed viability of tested K9BMMSCs.ConclusionPRP concentration plays an important role in K9BMMSCs viability, which could affect tissue repairs in vivo.
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Project description:Mesenchymal stem cells' (MSCs) fate is largely determined by the various topographical features and a range of extracellular matrix (ECM) components present in their niches. Apart from maintaining structural stability, they regulate cell morphology, division, proliferation, migration and differentiation among others. Traditional MSC cultures, which are mainly based on two-dimensional smooth surfaces of culture dishes and plates, do not provide topographical cues similar to in vivo three-dimensional niches, impacting various cellular processes. Therefore, we culture the mouse bone marrow-derived MSCs on microgrooved bearing surface, partially mimicking in vivo reticulated niche, to study its effect on morphology, pluripotency factor-associated stemness, cell division and rate of proliferation. Following culture, morphological features, and MSC-specific marker gene expression, such as CD29, CD44, Sca-1 along with HSC (Haematopoietic stem cell)-specific markers like CD34, CD45, CD11b were evaluated by microscopy and immunophenotyping, respectively. HSC is another type of bone marrow stem cell population, which concertedly interacts with MSC during various functions, including haematopoiesis. In addition, mesenchymal stem cells were further analyzed for gene expression of pluripotency-associated transcription factors such as Oct3/4, Sox-2, Nanog and Myc, as well as differentiated into adipocytes, osteocytes and chondrocytes. Our results show that microgrooved surface-cultured mesenchymal stem cells (MMSCs) expressed higher levels of expected cell surface and pluripotency-associated markers and proliferated more rapidly (2-3×fold) with higher percentage of cells in S/G2-M-phase, consequently giving rise to higher cell yield compared to standard culture flask-grown cells (MSCs), taken as control. Furthermore, both MSCs and MMSCs showed considerable accumulation of intracellular lipid-droplets, higher alkaline phosphatase activity and secretion of extracellular matrix that are characteristics of adipogenesis, osteogenesis and chondrogenesis, respectively.
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Project description:Bone marrow stromal cells (BMSCs) are capable of differentiating into multiple cell lineages including endothelial cells. Simvastatin, an HMG-CoA reductase inhibitor that is used as a cholesterol-lowering agent, promotes endothelial differentiation from epithelial progenitor cells (EPC). The Notch signaling pathway, which plays a key role in multiple cell functions such as differentiation, proliferation, and apoptosis, can be regulated by simvastatin. Therefore, we examined the effect of simvastatin on BMSC differentiation into endothelial cells and the underlying mechanisms involved in this process. We observed that simvastatin stimulation of rat BMSCs resulted in significantly increased expression of endothelial-specific genes and proteins, including von Willebrand factor (vWF), CD31, vascular endothelial-cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGFR2, Flk-1), and VEGF receptor 1 (VEGFR-1, Flt-1). Simvastatin also significantly increased capillary tubelike formation of the BMSCs. In addition, the intracellular cleavage of Notch (NICD) was markedly enhanced by simvastatin in BMSCs. Incubation of BMSCs with a gamma-secretase inhibitor, or Notch1 small interfering RNA (siRNA) that significantly inhibited the formation of NICD, blocked the expression of endothelial-specific markers in BMSCs and their differentiation into functional endothelial cells. These data suggest that simvastatin induces rat BMSCs differentiation into endothelial cells via a Notch signaling pathway.
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Project description:BackgroundBone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis.ResultsSamples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs.ConclusionOur results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.
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Project description:Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative (RT)PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions.
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Project description:Mesenchymal stem cells (MSCs) hold great potential in cell therapy and have attracted increasing interests in a wide range of biomedical sciences. However, the scarcity of MSCs and the prolonged isolation procedure limited the clinical application. To address these 2 issues, we developed a method to isolate MSCs from bone biopsy tissues of euthanized canine body donors. Compared to the traditional method to isolate MSCs from aspirated bone marrow (BMSCs), the isolation procedure for MSCs from harvested epiphyseal cancellous bone (EMSCs) was less time-consuming. The isolated EMSCs had similar plastic-adherence, tri-lineage differentiation and consistent surface marker profiles compared to BMSCs. We harvested BMSCs and EMSCs from 24 euthanized cases from clinics and 42 euthanized donors from a local shelter. The successful rate for EMSC isolation is significantly higher compared to BMSC isolation, while the other properties of the isolated MSCs including the clonogenicity, proliferative potentials and molecular phenotypes were not discernibly different between the MSCs established by the two methods. In conclusion, we demonstrated a new procedure to harvest MSCs by bone biopsy at the epiphyseal region. This method is less time consuming and more reliable, and the resulting MSCs are comparable to those harvested by bone marrow aspiration. The combination of the two methods can greatly improve the efficiency to harvest MSCs.
| S-EPMC4383772 | biostudies-literature
Project description:Background and aimMesenchymal stem cells (MSCs), which have multi-lineage differentiation potentials, are a promising source for regenerative medicine. However, the focus of study of MSCs is shifting from the characterization of the differentiation potential to their secretion potential for cell transplantation. Tissue regeneration and the attenuation of immune responses are thought to be affected by the secretion of multiple growth factors and cytokines by MSCs. However, the secretion potential of MSCs profiling remains incompletely characterized. In this study, we focused on the secretion ability related and protein mRNA expression of dog adipose tissue-derived MSCs (AT-MSC), bone marrow (BM)-derived MSCs, and BM-derived mononuclear cells (BM-MNC).Materials and methodsReal-time polymerase chain reaction analyses revealed mRNA expression of nine growth factors and seven interleukins in these types of cells and three growth factors protein expression were determined using Enzyme-linked immunosorbent assay.ResultsFor the BM-MNC growth factors, the mRNA expression of transforming growth factor-β (TGF-β) was the highest. For the BM-derived MSC (BM-MSC) and AT-MSC growth factors, the mRNA expression of vascular endothelial growth factor (VEGF) was highest. BM-MSCs and AT-MSCs showed similar expression profiles. In contrast, BM-MNCs showed unique expression profiles for hepatocyte growth factor and epidermal growth factor. The three types of cells showed a similar expression of TGF-β.ConclusionWe conclude that expression of cytokine proteins and mRNAs suggests involvement in tissue repair and protection.
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Project description:Fibroblast growth factor-2 delays the loss of chondrogenic potential in adult bone marrow-derived mesenchymal stem cells We compared human mesenchymal stem cells (hMSCs), expanded long-term with and without fibroblast growth factor (FGF) supplementation, with respect to their proliferation rate, and ability to differentiate along the chondrogenic pathway in vitro. hMSCs expanded in FGF-supplemented medium proliferated more rapidly than those expanded under control conditions. Aggregates of FGF-treated cells exhibited chondrogenic differentiation at passages 1 through 7, although, in some of the preparations, chondrogenic differentiation was somewhat diminished after seventh passage. Aggregates made with control cells differentiated along the chondrogenic lineage at first passage but exhibited only marginal chondrogenic differentiation after four passages and failed to form cartilage after seven passages. Microarray analysis of gene expression identified 334 transcripts that were differentially expressed in fourth passage control cells which had reduced chondrogenic potential compared to fourth passage FGF-treated cells which retained this differentiation capacity and 243 transcripts that were differentially expressed when comparing them to first passage control cells which were also capable of differentiating into chondrocytes. The intersection of these analyses yielded 49 transcripts that were differentially expressed in cells that exhibited chondrogenic differentiation in vitro compared to cells that did not. These preliminary data must now be validated to verify whether the different gene expression profiles translate into functional differences. These findings suggest that care should be exercised when extensively expanding these cells for cartilage tissue engineering applications. Keywords: time course and treatment
2009-10-29 | GSE14828 | GEO
Project description:Sarcoidosis is a devastating inflammatory disease affecting many organs, especially the lungs and lymph nodes. Bone marrow-derived mesenchymal stromal cells (MSCs) can "reprogram" various types of macrophages towards an anti-inflammatory phenotype. We wanted to determine whether alveolar macrophages from sarcoidosis subjects behave similarly by mounting an anti-inflammatory response when co-cultured with MSCs. Fifteen sarcoidosis and eight control subjects underwent bronchoscopy and bronchoalveolar lavage (BAL). Unselected BAL cells (70-94% macrophages) were isolated and cultured with and without MSCs from healthy adults. Following stimulation of the cultured cells with lipopolysaccharide, the medium was removed to measure interleukin 10 and tumor necrosis factor alpha (IL-10 and TNF-?). In two additional sarcoidosis subjects, flow cytometry was used to study intracellular cytokines and surface markers associated with alveolar macrophages to confirm the results. Unselected BAL cells from sarcoidosis subjects co-cultured with MSCs showed a reduction in TNF-? (pro-inflammatory M1) and an increase in IL-10 (anti-inflammatory M2) in 9 of 11 samples studied. Control subject samples showed few, if any, differences in cytokine production. Unselected BAL cells from two additional patients analyzed by flow cytometry confirmed a switch towards an anti-inflammatory state (i.e., M1 to M2) after co-culture with MSCs. These results suggest that, similarly to other macrophages, alveolar macrophages also respond to MSC contacts by changing towards an anti-inflammatory phenotype. Based on our results, we hypothesize that mesenchymal stromal cells applied to the airways might alleviate lung inflammation and decrease steroid need in patients with sarcoidosis.
| S-EPMC7019909 | biostudies-literature
Project description:Osteoporosis is a progressive systemic skeletal disease associated with decreased bone mineral density and deterioration of bone quality, and it affects millions of people worldwide. Currently, it is treated mainly using antiresorptive and osteoanabolic agents. However, these drugs have severe adverse effects. Cell replacement therapy using mesenchymal stem cells (MSCs) could serve as a treatment strategy for osteoporosis in the future. LIGHT (HVEM-L, TNFSF14, or CD258) is a member of the tumor necrosis factor superfamily. However, the effect of recombinant LIGHT (rhLIGHT) on osteogenesis in human bone marrow-derived MSCs (hBM-MSCs) is unknown. Therefore, we monitored the effects of LIGHT on osteogenesis of hBM-MSCs. Lymphotoxin-β receptor (LTβR), which is a LIGHT receptor, was constitutively expressed on the surface of hBM-MSCs. After rhLIGHT treatment, calcium and phosphate deposition in hBM-MSCs, stained by Alizarin red and von Kossa, respectively, significantly increased. We performed quantitative real-time polymerase chain reaction to examine the expressions of osteoprogenitor markers (RUNX2/CBFA1 and collagen I alpha 1) and osteoblast markers (alkaline phosphatase, osterix/Sp7, and osteocalcin) and immunoblotting to assess the underlying biological mechanisms following rhLIGHT treatment. We found that rhLIGHT treatment enhanced von Kossa- and Alizarin red-positive hBM-MSCs and induced the expression of diverse differentiation markers of osteogenesis in a dose-dependent manner. WNT/β-catenin pathway activation strongly mediated rhLIGHT-induced osteogenesis of hBM-MSCs, accelerating the differentiation of hBM-MSCs into osteocytes. In conclusion, the interaction between LIGHT and LTβR enhances osteogenesis of hBM-MSCs. Therefore, LIGHT might play an important role in stem cell therapy.
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