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Effective Osteogenic Priming of Mesenchymal Stem Cells through LNA-ASOs-Mediated Sfrp1 Gene Silencing.

ABSTRACT: Mesenchymal stem cell (MSC) transplantation has emerged as a promising approach for bone regeneration. Importantly, the beneficial effects of MSCs can be improved by modulating the expression levels of specific genes to stimulate MSC osteogenic differentiation. We have previously shown that Smurf1 silencing by using Locked Nucleic Acid-Antisense Oligonucleotides, in combination with a scaffold that sustainably releases low doses of BMP-2, was able to increase the osteogenic potential of MSCs in the presence of BMP-2 doses significantly smaller than those currently used in the clinic. This would potentially allow an important reduction in this protein in MSs-based treatments, and thus of the side effects linked to its administration. We have further improved this system by specifically targeting the Wnt pathway modulator Sfrp1. This approach not only increases MSC bone regeneration efficiency, but is also able to induce osteogenic differentiation in osteoporotic human MSCs, bypassing the need for BMP-2 induction, underscoring the regenerative potential of this system. Achieving successful osteogenesis with the sole use of LNA-ASOs, without the need of administering pro-osteogenic factors such as BMP-2, would not only reduce the cost of treatments, but would also open the possibility of targeting these LNA-ASOs specifically to MSCs in the bone marrow, allowing us to treat systemic bone loss such as that associated with osteoporosis.

SUBMITTER: Garcia-Sanchez D

PROVIDER: S-EPMC8398380 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS:hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis. RESULTS:Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS:Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.

Project description:BackgroundOsteoporosis affects the mandible resulting in bone loss. Though impairments are not life threatening, they affect a person's quality-of-life particularly vulnerable elderly. MicroRNAs (miRNAs) are novel regulatory factors that play an important role in regulating bone metabolism. Autophagy is evolutionarily conserved intracellular self-degradation process and is vital in the maintenance of both miRNA and bone homeostasis. However, the role of autophagy in the pathogenesis of miRNA regulating osteoporosis remains unclear.MethodsIn the study, we established a rat osteoporosis model induced by ovariectomy (OVX) and isolated mesenchymal stem cells from mandible (MMSCs-M). Several miRNAs were identified to regulate osteoporosis in some studies. qRT-PCR was applied to examine the expression of miRNA, autophagy and osteogenic differentiation-related genes. Western blotting assays were performed to detect the expression of autophagy and osteogenic differentiation proteins. Immunofluorescence and transmission electron microscope were used to verify the autophagy activity. Transfecting technology was used to enhance or suppress the expression of miR-152-5p which enable us to observe the relationship between miR-152-5p, autophagy and osteogenic differentiation. Additionally, the measurement of reactive oxygen species was used to investigate the mechanism of autophagy affecting osteogenic differentiation.ResultsWe found an upregulated expression of miR-152-5p in MMSCs-M in OVX group. Downregulated autophagy-related gene, proteins and autophagosome were detected in vitro of OVX group compared with sham group. Moreover, downregulation of miR-152-5p promoted osteogenic differentiation of MMSCs-M as well as enhanced autophagy-related proteins in OVX group. Conversely, overexpression of miR-152-5p showed opposite effect in sham group. Meanwhile, we found Atg14 (autophagy-related protein homolog 14) was identified to be a direct target of miR-152-5p theoretically and functionally. In other words, we confirmed inhibition of miR-152-5p promoted the osteogenic differentiation via promoting ATG14-mediated autophagy. Furthermore, miR-152-5p/ATG14-mediated autophagy regulated osteogenic differentiation by reducing the endogenous ROS accumulation and maintaining cellular redox homeostasis.ConclusionOur data suggest that miR-152-5p is the first identified to regulate osteogenic differentiation by directly targeting autophagy-related protein ATG14 and regulating oxidative stress and therapeutic inhibition of miR-152-5p may be an efficient anabolic strategy for osteoporosis.

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Effective Osteogenic Priming of Mesenchymal Stem Cells through LNA-ASOs-Mediated Sfrp1 Gene Silencing.

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