scholarly journals Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Neety Sahu ◽  
Gaurav Budhiraja ◽  
Anuradha Subramanian
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Signaling Pathways ◽  
Mesenchymal Stromal Cells ◽  
Nuclear Localization ◽  
Stromal Cells ◽  
Actin Reorganization ◽  
Low Intensity ◽  
Gene And Protein Expression ◽  
Low Intensity Ultrasound

Abstract Background Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS.

scholarly journals Interleukin-1β Induced Matrix Metalloproteinase Expression in Human Periodontal Ligament-Derived Mesenchymal Stromal Cells under In Vitro Simulated Static Orthodontic Forces

2021 ◽  
Vol 22 (3) ◽  
pp. 1027
Author(s):  
Christian Behm ◽  
Michael Nemec ◽  
Alice Blufstein ◽  
Maria Schubert ◽  
Xiaohui Rausch-Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Periodontal Ligament ◽  
Induced Expression ◽  
Gene And Protein Expression ◽  
Tensile Strains ◽  
Orthodontic Forces ◽  
Low Magnitude

The periodontal ligament (PDL) responds to applied orthodontic forces by extracellular matrix (ECM) remodeling, in which human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) are largely involved by producing matrix metalloproteinases (MMPs) and their local inhibitors (TIMPs). Apart from orthodontic forces, the synthesis of MMPs and TIMPs is influenced by the aseptic inflammation occurring during orthodontic treatment. Interleukin (IL)-1β is one of the most abundant inflammatory mediators in this process and crucially affects the expression of MMPs and TIMPs in the presence of cyclic low-magnitude orthodontic tensile forces. In this study we aimed to investigate, for the first time, how IL-1β induced expression of MMPs, TIMPs and how IL-1β in hPDL-MSCs was changed after applying in vitro low-magnitude orthodontic tensile strains in a static application mode. Hence, primary hPDL-MSCs were stimulated with IL-1β in combination with static tensile strains (STS) with 6% elongation. After 6- and 24 h, MMP-1, MMP-2, TIMP-1 and IL-1β expression levels were measured. STS alone had no influence on the basal expression of investigated target genes, whereas IL-1β caused increased expression of these genes. In combination, they increased the gene and protein expression of MMP-1 and the gene expression of MMP-2 after 24 h. After 6 h, STS reduced IL-1β-induced MMP-1 synthesis and MMP-2 gene expression. IL-1β-induced TIMP-1 gene expression was decreased by STS after 6- and 24-h. At both time points, the IL-1β-induced gene expression of IL-1β was increased. Additionally, this study showed that fetal bovine serum (FBS) caused an overall suppression of IL-1β-induced expression of MMP-1, MMP-2 and TIMP-1. Further, it caused lower or opposite effects of STS on IL-1β-induced expression. These observations suggest that low-magnitude orthodontic tensile strains may favor a more inflammatory and destructive response of hPDL-MSCs when using a static application form and that this response is highly influenced by the presence of FBS in vitro.

scholarly journals Continuous Low-Intensity Ultrasound Preserves Mitochondrial Potential, Inhibits NFκB Activation and Rescues Chondrogenesis of Mesenchymal Stromal Cells

2021 ◽  
Author(s):  
Sarayu Bhogoju ◽  
Shahid Khan ◽  
Denzhi Wang ◽  
Anuradha Subramanian
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Cartilage Repair ◽  
Stromal Cells ◽  
Nuclear Translocation ◽  
Feedback Mechanism ◽  
Protective Agent ◽  
Mitochondrial Potential ◽  
Qrt Pcr ◽  
Low Intensity ◽  
Low Intensity Ultrasound

Abstract Objective: Dysregulation of the anabolic processes in a proinflammatory joint environment coupled with impeded chondrogenic differentiation of mesenchymal stromal cells (MSCs) led to inferior cartilage repair outcomes. The preponderance of proinflammatory cytokines activated nuclear factor kappa B (NFκB) and impeded the chondrogenesis of MSCs. Thus, strategies that minimize the deleterious effects of activated NFκB while promoting MSC chondrogenesis are of interest. The present study establishes the ability of continuous low-intensity ultrasound (cLIUS) to rescue MSC chondrogenesis impacted by a proinflammatory environment. Methods: Human bone marrow-derived MSCs were seeded in alginate:collagen hydrogels and cultured for 21-days in an ultrasound-assisted bioreactor 14 kPa (5.0 MHz, 2.5 Vpp; 4-applications/day) for 21 days in the presence of IL1β and evaluated by qRT-PCR (n=10), immunofluorescence (n=15), western blotting (WB) (n=6), and immunohistochemistry (n=3). The differential expression of markers associated with NFκB pathway under cLIUS were evaluated upon a single exposure of cLIUS and assayed by qRT-PCR (n=3), immunofluorescence (n=30-60), WB (n=6) and tetramethylrhodamine methyl ester assay (n=50) was used to assess the mitochondrial potential under IL1β and cLIUS treatment.Results: Chondroinductive potential of cLIUS was preserved as noted by the increased expression of SOX9 and deposition of collagen II. cLIUS extended its chondroprotective effects by stabilizing the NFκB complex in the cytoplasm via engaging the IκBα feedback mechanism, thus preventing its nuclear translocation. cLIUS acted as a mitochondrial protective agent by restoring the mitochondrial potential and the mitochondrial mRNA expression in a proinflammatory environment. Conclusion: Our results demonstrated the potential of cLIUS for cartilage repair and regeneration under proinflammatory conditions.

Mesenchymal Stromal Cells From Myelodysplastic Syndrome Patients Show Lower DICER1 and DROSHA Expression, as Well as Decreased Mir-155 and Mir-181a MicroRNA Expression, When Compared with Healthy Controls

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 397-397
Author(s):  
Carlos Santamaría ◽  
Olga López-VIllar ◽  
Sandra Muntión ◽  
Belén Blanco ◽  
Soraya Carrancio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Protein Expression ◽  
Myelodysplastic Syndrome ◽  
Western Blot ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Microrna Expression ◽  
Healthy Controls ◽  
Lower Expression

Abstract Abstract 397 Mesenchymal stromal cells (MSC) are closely related to the regulation of hematopoietic stem cell niche. Recently, Raaijmakers et al (Nature, 2010), published that deletion of Dicer1, a RNase III enzyme involved in microRNA biogenesis, in murine MSC-derived osteoprogenitors triggered peripherical blood cytopenias, myelodysplasia and subsequent AML, showing that molecular alterations in bone marrow microenvironment could result in clonal impaired haematopoiesis. Here, we have investigated whether MSC from myelodysplastic syndrome (MDS) patients show differences in DICER1 and DROSHA, another RNA III endonuclease, in comparison to healthy MSC. In addition, we have analyzed several hematopoietic-related microRNAs in these same samples. Bone marrow MSC from MDS patients (n=35; 10 5q- syndrome, 4 RA, 5 RARS, 10 RCMD, 3 RAEB, 2 MDS-U and 1 hypocellular MDS) and healthy donors (HD, n=20) were isolated and in vitro expanded following standard procedures until the third passage. Additionally, paired mononuclear cells (MNC) from 13 MDS and 8 HD were obtained. Total RNA was isolated using TRIzol reagent (Invitrogen). DICER1 and DROSHA relative gene expressions were assessed by quantitative PCR (Q-PCR) using commercial TaqMan® assay (Applied Biosystems®) with GAPDH as control gene. DICER1 and DROSHA (Abcam) protein expression were evaluated in whole cell lysates by western blot, using calnexin (Stressgen) as control. Several microRNAs with known role in hematopoiesis and immune system regulation were analyzed in 25 MDS and 12 HD by Q-PCR using commercial TaqMan® MicroRNA assay (Applied Biosystems®) with RNU43 as control microRNA. MSC from MDS showed significant lower DICER1 (0.0035±0.0020 vs. 0.0076±0.0092; p=0.044) and DROSHA (0.0070±0.0028 vs. 0.0135±0.0176; p=0.019) gene expression levels than healthy controls. Moreover, MSC from MDS showed lower protein expression of both DICER1 and DROSHA by western blot analysis, confirming Q-PCR findings. By contrast, no difference in either DICER1 (0.0197±0.0151 vs. 0.0173±0.0112; p=0.9) or DROSHA (0.0089±0.0023 vs. 0.0067±0.0037; p=0.09) gene expression were observed between MNC from MDS and HD. As far as microRNA expression, we observed a lower expression of mir-155 (0.63±0.92 vs. 0.94±0.49; p=0.007) and mir-181a (1.30±0.95 vs. 2.02±1.05; p=0.041) in MSC from MDS in comparison to healthy controls. Mir-155 and mir-181a are involved in T-cell and B-cell differentiation, while mir-155 are also related to erythroid and megakarycytic differentiation. We conclude that MSC from MDS patients show lower expression of DICER and DROSHA, two relevant RNA-III endonucleases involved in the microRNA biogenesis, confirming recent findings in murine models. Moreover, the expression of some microRNA is impaired in these cells, raising the possibility that these microenvironmental alterations could be involved in the MDS pathophysiology. Disclosures: No relevant conflicts of interest to declare.

Inducible Levels of Gelatinase B/Matrix Metalloproteinase-9 Gene Expression in Monocytes Are Associated with Marrow Cellularity in Myelodysplastic Syndrome (MDS).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1391-1391
Author(s):  
Mineo Iwata ◽  
Manoj Pillai ◽  
H. Joachim Deeg ◽  
Ghislain Opdenakker ◽  
Beverly Torok-Storb
Keyword(s):  
Gene Expression ◽  
Matrix Metalloproteinase ◽  
Protein Expression ◽  
Myelodysplastic Syndrome ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Matrix Metalloproteinase 9 ◽  
Gene And Protein Expression ◽  
Marrow Cellularity ◽  
Metalloproteinase 9

Abstract Evidence suggests that within the hematopoietic microenvironment (ME) stromal cell function can be modified by activities produced by monocytes/macrophages and that the reciprocal is also true; stroma can influence monocyte function. Critical regulatory molecules produced by stroma are often membrane bound until cleaved by metalloproteinases (MMP); cleavage can serve to either activate or inactivate their functions, making MMPs critical components of hematopoietic regulation. We report here that gene and protein expression of human matrix metalloproteinase-9 (MMP-9) is induced in monocytes in vitro by stromal cell conditioned media (CM). Briefly, flow sorted CD14+ peripheral blood monocytes were cultured for 5 days in the presence or absence of CM from HS-5 stromal cells, and MMP-9 gene expression was determined by real time PCR. Little or no MMP-9 gene expression was detected in CD14+ cells on day 0 prior to culture. In contrast, after 5 days of culture in control media MMP-9 gene expression was increased significantly (p=0.02). However culturing CD14+ cells in CM significantly increased expression another 3 fold (p<0.0001). MMP-9 protein secretion was also increased 12-fold after culture in CM. To identify which factors participate in the induction of MMP-9, the levels of MMP-9 mRNA were determined after CD14+ cells were cultured for 5 days in the presence or absence of 9 different recombinant factors. Factors chosen were those known to be present in CM and known to have their receptors expressed by monocytes. MMP-9 gene and protein expression increased 4 to 8-fold with 0.5ng/mL MCP-1/CCL2 or IL-1b, other factors tested had negligible effects. Immune cytochemical localization of MMP-9 protein in bone marrow biopsies from healthy donors and patients with myelodysplastic syndrome (MDS) indicated that mature myeloid cells including granulocytes and monocytes were stained strongly for MMP-9 protein, whereas stromal cells, fat cells, megakaryocytes, immature myeloid cells, blasts and cells of the erythroid lineage were negative. While levels of CM-induced MMP-9 gene expression among monocytes from 10 normal donors were relatively consistent, there was significant variation in inducible gene expression among CD14+ cells from 25 MDS patients (p= 0.02). Further analysis of MMP-9 gene expression in MDS mononuclear cells indicated that induced levels were negatively correlated with the degree of marrow cellularity (p=0.0002). Although marrow cellularity is a subjective estimate that can vary from one area of bone to another, the strong statistical correlation obtained suggests it may be related directly or indirectly to MMP-9 levels. In conclusion, monocytes can express and secrete MMP-9 in response to factors secreted by stromal cells. We hypothesize that the response of MDS monocytes to stromal signals can be abnormal resulting in unusually high or low levels of MMP-9, and that this may, directly or indirectly, influence marrow cellularity in MDS patients.

scholarly journals Beneficial Role of Low-Intensity Laser Irradiation on Neural β-tubulin III Protein Expression in Human Bone Marrow Multipotent Mesenchymal Stromal Cells

2018 ◽  
Vol 14 (4) ◽  
pp. 585-598 ◽  
Author(s):  
Valéria Ferreira-Silva ◽  
Fernando L. Primo ◽  
Munira M.A. Baqui ◽  
Danielle A.R. Magalhães ◽  
Maristela D. Orellana ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Expression ◽  
Laser Irradiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Low Intensity ◽  
Intensity Laser ◽  
Β Tubulin

scholarly journals Alterations of mesenchymal stromal cells in cerebrospinal fluid: insights from transcriptomics and an ALS clinical trial

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ashley A. Krull ◽  
Deborah O. Setter ◽  
Tania F. Gendron ◽  
Sybil C. L. Hrstka ◽  
Michael J. Polzin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cerebrospinal Fluid ◽  
Growth Factors ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Large Scale ◽  
Therapeutic Benefit ◽  
Protein Levels ◽  
Genes Encoding ◽  
Intrathecal Space

Abstract Background Mesenchymal stromal cells (MSCs) have been studied with increasing intensity as clinicians and researchers strive to understand the ability of MSCs to modulate disease progression and promote tissue regeneration. As MSCs are used for diverse applications, it is important to appreciate how specific physiological environments may stimulate changes that alter the phenotype of the cells. One need for neuroregenerative applications is to characterize the spectrum of MSC responses to the cerebrospinal fluid (CSF) environment after their injection into the intrathecal space. Mechanistic understanding of cellular biology in response to the CSF environment may predict the ability of MSCs to promote injury repair or provide neuroprotection in neurodegenerative diseases. Methods In this study, we characterized changes in morphology, metabolism, and gene expression occurring in human adipose-derived MSCs cultured in human (hCSF) or artificial CSF (aCSF) as well as examined relevant protein levels in the CSF of subjects treated with MSCs for amyotrophic lateral sclerosis (ALS). Results Our results demonstrated that, under intrathecal-like conditions, MSCs retained their morphology, though they became quiescent. Large-scale transcriptomic analysis of MSCs revealed a distinct gene expression profile for cells cultured in aCSF. The aCSF culture environment induced expression of genes related to angiogenesis and immunomodulation. In addition, MSCs in aCSF expressed genes encoding nutritional growth factors to expression levels at or above those of control cells. Furthermore, we observed a dose-dependent increase in growth factors and immunomodulatory cytokines in CSF from subjects with ALS treated intrathecally with autologous MSCs. Conclusions Overall, our results suggest that MSCs injected into the intrathecal space in ongoing clinical trials remain viable and may provide a therapeutic benefit to patients.

scholarly journals Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Matthew Mannarino ◽  
Hosni Cherif ◽  
Li Li ◽  
Kai Sheng ◽  
Oded Rabau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Back Pain ◽  
Protein Expression ◽  
Disc Degeneration ◽  
Cell Senescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Matrix Synthesis ◽  
Gene And Protein Expression ◽  
Pain Patients ◽  
In Cells

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

scholarly journals Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile

2015 ◽  
Vol 24 (23) ◽  
pp. 2822-2840 ◽  
Author(s):  
Lindolfo da Silva Meirelles ◽  
Tathiane Maistro Malta ◽  
Virgínia Mara de Deus Wagatsuma ◽  
Patrícia Viana Bonini Palma ◽  
Amélia Goes Araújo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Gene Expression Profile ◽  
Mesenchymal Stromal Cells ◽  
Expression Profile ◽  
Stromal Cells ◽  
Human Adipose Tissue ◽  
Similar Gene Expression Profile ◽  
Similar Gene

scholarly journals Shp2 in uterine stromal cells critically regulates on time embryo implantation and stromal decidualization by multiple pathways during early pregnancy

PLoS Genetics ◽  
2022 ◽  
Vol 18 (1) ◽  
pp. e1010018
Author(s):  
Jianghong Cheng ◽  
Jia Liang ◽  
Yingzhe Li ◽  
Xia Gao ◽  
Mengjun Ji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Stromal Cells ◽  
Embryo Implantation ◽  
Endometrial Stromal Cells ◽  
Conditional Deletion ◽  
Enhancer Binding Protein ◽  
Epithelial Remodeling ◽  
Multiple Signaling ◽  
Transcription 3

Approximately 75% of failed pregnancies are considered to be due to embryo implantation failure or defects. Nevertheless, the explicit signaling mechanisms governing this process have not yet been elucidated. Here, we found that conditional deletion of the Shp2 gene in mouse uterine stromal cells deferred embryo implantation and inhibited the decidualization of stromal cells, which led to embryonic developmental delay and to the death of numerous embryos mid-gestation, ultimately reducing female fertility. The absence of Shp2 in stromal cells increased the proliferation of endometrial epithelial cells, thereby disturbing endometrial epithelial remodeling. However, Shp2 deletion impaired the proliferation and polyploidization of stromal cells, which are distinct characteristics of decidualization. In human endometrial stromal cells (hESCs), Shp2 expression gradually increased during the decidualization process. Knockout of Shp2 blocked the decidual differentiation of hESCs, while Shp2 overexpression had the opposite effect. Shp2 knockout inhibited the proliferation of hESCs during decidualization. Whole gene expression profiling analysis of hESCs during the decidualization process showed that Shp2 deficiency disrupted many signaling transduction pathways and gene expression. Analyses of hESCs and mouse uterine tissues confirmed that the signaling pathways extracellular regulated protein kinases (ERK), protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3) and their downstream transcription factors CCAAT/enhancer binding protein β (C/EBPβ) and Forkhead box transcription factor O1 (FOXO-1) were involved in the Shp2 regulation of decidualization. In summary, these results demonstrate that Shp2 plays a crucial role in stromal decidualization by mediating and coordinating multiple signaling pathways in uterine stromal cells. Our discovery possibly provides a novel key regulator of embryo implantation and novel therapeutic target for pregnancy failure.

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