Effect of high cyclic hydrostatic pressure on osteogenesis of mesenchymal stem cells cultured in liquefied micro-compartments
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ABSTRACT:
SUBMITTER: Ghasemzadeh-Hasankolaei M
PROVIDER: S-EPMC10696388 | biostudies-literature | 2023 Nov
REPOSITORIES: biostudies-literature
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Project description:Mesenchymal stem cells (MSCs) are known to be ideal candidates for clinical applications where not only regenerative potential but also immunomodulation ability is fundamental. Over the last years, increasing efforts have been put into the design and fabrication of 3D synthetic niches, conceived to emulate the native tissue microenvironment and aiming at efficiently controlling the MSC phenotype in vitro. In this panorama, our group patented an engineered microstructured scaffold, called Nichoid. It is fabricated through two-photon polymerization, a technique enabling the creation of 3D structures with control of scaffold geometry at the cell level and spatial resolution beyond the diffraction limit, down to 100 nm. The Nichoid's capacity to maintain higher levels of stemness as compared to 2D substrates, with no need for adding exogenous soluble factors, has already been demonstrated in MSCs, neural precursors, and murine embryonic stem cells. In this work, we evaluated how three-dimensionality can influence the whole gene expression profile in rat MSCs. Our results show that at only 4 days from cell seeding, gene activation is affected in a significant way, since 654 genes appear to be differentially expressed (392 upregulated and 262 downregulated) between cells cultured in 3D Nichoids and in 2D controls. The functional enrichment analysis shows that differentially expressed genes are mainly enriched in pathways related to the actin cytoskeleton, extracellular matrix (ECM), and, in particular, cell adhesion molecules (CAMs), thus confirming the important role of cell morphology and adhesions in determining the MSC phenotype. In conclusion, our results suggest that the Nichoid, thanks to its exclusive architecture and 3D cell adhesion properties, is not only a useful tool for governing cell stemness but could also be a means for controlling immune-related MSC features specifically involved in cell migration.
| S-EPMC9852851 | biostudies-literature
Project description:We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over 47,000 gene probes. Human MSC were seeded in three-dimensional collagen type I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and wingless-type MMTV integration site family, member 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain 4 (PDLIM4) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population.
| S-EPMC3875187 | biostudies-literature
Project description:BackgroundPhysical loading is necessary to maintain bone tissue integrity. Loading-induced fluid shear is recognised as one of the most potent bone micromechanical cues and has been shown to direct stem cell osteogenesis. However, the effect of pressure transients, which drive fluid flow, on human bone marrow stem cell (hBMSC) osteogenesis is undetermined. Therefore, the objective of the study is to employ a systematic analysis of cyclic hydrostatic pressure (CHP) parameters predicted to occur in vivo on early hBMSC osteogenic responses and late-stage osteogenic lineage commitment.MethodshBMSC were exposed to CHP of 10 kPa, 100 kPa and 300 kPa magnitudes at frequencies of 0.5 Hz, 1 Hz and 2 Hz for 1 h, 2 h and 4 h of stimulation, and the effect on early osteogenic gene expression of COX2, RUNX2 and OPN was determined. Moreover, to decipher whether CHP can induce stem cell lineage commitment, hBMSCs were stimulated for 4 days for 2 h/day using 10 kPa, 100 kPa and 300 kPa pressures at 2 Hz frequency and cultured statically for an additional 1-2 weeks. Pressure-induced osteogenesis was quantified based on ATP release, collagen synthesis and mineral deposition.ResultsCHP elicited a positive, but variable, early osteogenic response in hBMSCs in a magnitude- and frequency-dependent manner, that is gene specific. COX2 expression elicited magnitude-dependent effects which were not present for RUNX2 or OPN mRNA expression. However, the most robust pro-osteogenic response was found at the highest magnitude (300 kPa) and frequency regimes (2 Hz). Interestingly, long-term mechanical stimulation utilising 2 Hz frequency elicited a magnitude-dependent release of ATP; however, all magnitudes promoted similar levels of collagen synthesis and significant mineral deposition, demonstrating that lineage commitment is magnitude independent. This therefore demonstrates that physiological levels of pressures, as low as 10 kPa, within the bone can drive hBMSC osteogenic lineage commitment.ConclusionOverall, these findings demonstrate an important role for cyclic hydrostatic pressure in hBMSCs and bone mechanobiology, which should be considered when studying pressure-driven fluid shear effects in hBMSCs mechanobiology. Moreover, these findings may have clinical implication in terms of bioreactor-based bone tissue engineering strategies.
| S-EPMC6203194 | biostudies-literature
Project description:While yes-associated protein (YAP) is now recognized as a potent mechanosensitive transcriptional regulator to affect cell growth and differentiation including the osteogenic transcription of mesenchymal stem cells (MSCs), most studies have reported the YAP mechanosensing of static mechanophysical cues such as substrate stiffness. We tested MSC response to dynamic loading, i.e., cyclic mechanical stretching, and assessed YAP mechanosensing and resultant MSC osteogenesis. We showed that cyclic stretching at 10% strain and 1 Hz frequency triggered YAP nuclear import in MSCs. YAP phosphorylation at S127 and S397, which is required for YAP cytoplasmic retention, was suppressed by cyclic stretch. We also observed that anti-YAP-regulatory Hippo pathway, LATS phosphorylation, was significantly decreased by stretch. We confirmed the stretch induction of MSC osteogenic transcription and differentiation, and this was impaired under YAP siRNA suggesting a key role of YAP dynamic mechanosensing in MSC osteogenesis. As an underlying mechanism, we showed that the YAP nuclear transport by cyclic stretch was abrogated by ROCK inhibitor, Y27632. ROCK inhibitor also impaired the stretch induction of F-actin formation and MSC osteogenesis, thus implicating the role of the ROCK-F-actin cascade in stretch-YAP dynamic mechanosensing-MSC osteogenesis. Our results provide insight into bone tissue engineering and skeletal regenerative capacity of MSCs especially as regards the role of dynamic mechanical loading control of YAP-mediated MSC osteogenic transcription.
| S-EPMC10809151 | biostudies-literature
Project description:It has been previously demonstrated that uniaxial cyclic stretching (UCS) induces differentiation of mesenchymal stem cells (MSCs) into osteoblasts in vitro. It is also known that interactions between cells and external forces occur at various aspects including cell-matrix, cytoskeleton, nucleus membrane, and chromatin. However, changes in chromatin landscape during this process are still not clear. The present study was aimed to determine changes of chromatin accessibility under cyclic stretch. The influence of cyclic stretching on the morphology, proliferation, and differentiation of hMSCs was characterized. Changes of open chromatin sites were determined by assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq). Our results showed that UCS induced cell reorientation and actin stress fibers realignment, and in turn caused nuclear reorientation and deformation. Compared with unstrained group, the expression of osteogenic and chondrogenic marker genes were the highest in group of 1 Hz + 8% strain; this condition also led to lower cell proliferation rate. Furthermore, there were 2022 gene loci with upregulated chromatin accessibility in 1 Hz + 8% groups based on the analysis of chromatin accessibility. These genes are associated with regulation of cell morphogenesis, cell-substrate adhesion, and ossification. Signaling pathways involved in osteogenic differentiation were found in up-regulated GO biological processes. These findings demonstrated that UCS increased the openness of gene loci associated with regulation of cell morphogenesis and osteogenesis as well as the corresponding transcription activities. Moreover, the findings also connect the changes in chromatin accessibility with cell reorientation, nuclear reorientation, and deformation. Our study may provide reference for directed differentiation of stem cells induced by mechanical microenvironments.
| S-EPMC8294092 | biostudies-literature
Project description:This study addresses the hypothesis that renal interstitial (RI) cGMP, a modulator of pressure-natriuresis, exerts its effect through a relationship with renal interstitial hydrostatic pressure (RIHP). Increasing renal perfusion pressure in Sprague-Dawley rats led to increases in RIHP (5.2+/-0.6 to 10.9+/-1.6 mm Hg; P<0.01), urine sodium excretion (0.062+/-0.009 to 0.420+/-0.068 micromol/min per gram; P<0.01), and RI cGMP (3.5+/-0.8 to 9.5+/-1.7 fmol/min; P<0.01), and these effects were blocked by partial renal decapsulation. Infusion of cGMP into the RI compartment of decapsulated animals restored natriuresis (0.067+/-0.010 to 0.310+/-0.061 micromol/min per gram; P<0.01). These changes were independent of changes in glomerular filtration rate . Artificially increasing RIHP in normotensive animals increased RI cGMP (4.1+/-0.6 to 6.9+/-0.7 fmol/min; P<0.01) and urine sodium excretion (0.071+/-0.013 to 0.179+/-0.039 micromol/min per gram; P<0.05). Coinfusion of organic anion transport-inhibitor probenecid, or soluble guanylyl cyclase inhibitor 1-H(1,2,4) oxadiazolo-(4,2)quinoxalin-1-one, abolished these effects. Infusion of cGMP into the RI compartment of normotensive animals increased RIHP (6.7+/-0.4 to 10.3+/-0.9 mm Hg; P<0.001). Exogenous RI cGMP delivery did not affect total, cortical, or medullary renal blood flow. These studies suggest that extracellular RI cGMP is required for the natriuresis observed after increases in renal perfusion pressure and RIHP and that cGMP acts via a tubule mechanism. The results support an intrarenal positive-feedback loop wherein RI cGMP increases RIHP, which, in turn, increases RI cGMP, contributing to the reinforcement of pressure-natriuresis.
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Project description:Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38-, and CD133+CD38-) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems.
| S-EPMC5848107 | biostudies-literature
Project description:S. cerevisiae Y440 Mat a leu2 was grown in YEPD at 28 degrees C with aeration to exponential growth phase and was subjected to a hydrostatic pressure of 50 and 200 MPa for 30 minutes at room temperature. Total RNA was extracted using phenol/chloroform and further precipitated with 3 M sodium acetate / absolute ethanol. Extracted RNA samples were treated for 10 min with 0.5 U of RNAse-free DNAse I / ]g RNA at 37 oC to remove any residual genomic DNA. RNA pellets were washed in 70 % ethanol and resuspended in DEPC treated water. Purified mRNA from pressurized and unpressurized cells was reversed-transcribed, labeled with fluorescent-tagged nucleotides, and hybridized against a common refernce pool of mRNA for 18 h at 65 0C on cDNA microarray. After several washes, arrays were scanned using a commercially available scanning laser microscope (GenePix 4000) from Axon Instruments (Foster City, CA), the data obtained was normalized (mean value) applying a linear regression method. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract Keywords: Computed
2005-12-30 | GSE3935 | GEO
Project description:Osteogenesis imperfecta (OI) is a disease caused by mutations in different genes resulting in mild, severe, or lethal forms. With no cure, researchers have investigated the use of cell therapy to correct the underlying molecular defects of OI. Mesenchymal stem cells (MSCs) are of particular interest because of their differentiation capacity, immunomodulatory effects, and their ability to migrate to sites of damage. MSCs can be isolated from different sources, expanded in culture, and have been shown to be safe in numerous clinical applications. This review summarizes the preclinical and clinical studies of MSCs in the treatment of OI. Altogether, the culmination of these studies show that MSCs from different sources: 1) are safe to use in the clinic, 2) migrate to fracture sites and growth sites in bone, 3) engraft in low levels, 4) improve clinical outcome but have a transient effect, 5) have a therapeutic effect most likely due to paracrine mechanisms, and 6) have a reduced therapeutic potential when isolated from patients with OI.
| S-EPMC9892307 | biostudies-literature
Project description:Our results showed that UCS induced cell reorientation and actin fibers realignment, and in turn caused nuclear reorientation and deformation. Furthermore, UCS promoted the expression of osteogenic and chondrogenic marker genes. Based on assay for transposase accessible chromatin with high throughput sequencing (ATAC-seq), gene loci with up-regulated chromatin accessibility under UCS were found associate with regulation of cell morphogenesis, ossification, and osteogenic differentiation signaling pathways. These findings demonstrated UCS increased the openness of gene loci associated with cell morphogenesis and osteogenesis as well as the corresponding transcription activities. Moreover, our findings also connect the changes in chromatin accessibility with cell reorientation, nuclear reorientation and deformation.
2021-07-28 | GSE166410 | GEO