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Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

ABSTRACT: Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

SUBMITTER: Chamberlain CS

PROVIDER: S-EPMC11228621 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Chamberlain Connie S CS Prabahar Archana A Kink John A JA Mueller Erika E Li Yiyao Y Yopp Stephanie S Capitini Christian M CM Hematti Peiman P Murphy William L WL Vanderby Ray R Jiang Peng P

Stem cells (Dayton, Ohio) 20240701 7

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in ...[more]

PMID: 38597671

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Project description:IntroductionMesenchymal stromal cells (MSCs) play a crucial role in promoting tissue regeneration and healing, particularly in bone tissue. Both smoking and nicotine use are known to delay and inhibit the healing process in patients. This study aims at delineating these cellular effects by comparing the impact of nicotine alone to cigarette smoke with equivalent nicotine content, and shedding light on potential differences in the healing process.MethodsWe examined how cigarette smoke and nicotine affect the migration, proliferation, and osteogenic differentiation of human patient-derived MSCs in vitro, as well as the secretion of cytokines IL-6 and IL-8. We measured nicotine concentration of the cigarette smoke extract (CSE) to clarify the role of the nicotine in the effect of the cigarette smoke.ResultsMSCs exposed to nicotine-concentration-standardized CSE exhibited impaired wound healing capability, and at high concentrations, increased cell death. At lower concentrations, CSE dose-dependently impaired migration, proliferation, and osteogenic differentiation, and increased IL-8 secretion. Nicotine impaired proliferation and decreased PINP secretion. While there was a trend for elevated IL-6 levels by nicotine in undifferentiated MSCs, these changes were not statistically significant. Exposure of MSCs to equivalent concentrations of nicotine consistently elicited stronger responses by CSE and had a more pronounced effect on all studied parameters. Our results suggest that the direct effect of cigarette smoke on MSCs contributes to impaired MSC function, that adds to the nicotine effects.ConclusionsCigarette smoke extract reduced the migration, proliferation, and osteogenic differentiation in MSCs in vitro, while nicotine alone reduced proliferation. Cigarette smoke impairs the osteogenic and regenerative ability of MSCs in a direct cytotoxic manner. Cytotoxic effect of nicotine alone impairs regenerative ability of MSCs, but it only partly explains cytotoxic effects of cigarette smoke. Direct effect of cigarette smoke, and partly nicotine, on MSCs could contribute to the smoking-related negative impact on long-term bone health, especially in bone healing.

Project description:BackgroundThe anterior cruciate ligament (ACL) fails to heal after rupture, leading to joint instability and an increased risk of osteoarthritis. Mesenchymal stem/stromal cell (MSC) exosomes have reported wide-ranging therapeutic efficacy; however, their potential for augmenting ACL repair remains to be investigated.PurposeTo evaluate the use of MSC exosomes with fibrin sealant on biological augmentation of ACL healing after suture repair and their effects on ACL fibroblast functions.Study designControlled laboratory study.MethodsTwelve rabbit knees underwent ACL transection and suture repair. MSC exosome and fibrin composite (Exosome+Fibrin) or fibrin (Fibrin) alone was used to supplement the suture repair in 6 knees. ACL repair was assessed by magnetic resonance imaging at 6 and 12 weeks postoperatively and by histologic and immunohistochemical analyses at 12 weeks. To investigate the mechanisms through which MSC exosomes augment ACL repair, metabolic activity, proliferation, migration, and matrix synthesis assays were performed using the primary ACL fibroblasts. RNA sequencing was also performed to assess global gene expression changes in exosome-treated ACL fibroblasts.ResultsBased on magnetic resonance imaging findings, 5 of 6 Exosome+Fibrin-treated ACLs were completely or partially healed, as opposed to 5 of 6 Fibrin-treated ACLs appearing torn at 6 and 12 weeks postoperatively. Additionally, 4 of 6 Exosome+Fibrin-treated ACLs were isointense, as compared with 5 of 6 Fibrin-treated ACLs that were hyperintense, indicating improved remodeling and maturation of the repaired ACLs with Exosome+Fibrin treatment. Histologically, Exosome+Fibrin-treated ACLs showed more organized collagen fibers and abundant collagen deposition, with a high amount of collagen I and relatively lower amount of collagen III, which are consistent with the matrix structure and composition of the normal ACL. Cell culture studies using ACL fibroblasts showed that MSC exosomes enhanced proliferation, migration, and collagen synthesis and deposition, which are cellular processes relevant to ACL repair. Further gene set enrichment analysis revealed key pathways mediated by MSC exosomes in enhancing proliferation and migration while reducing matrix degradation of ACL fibroblasts.ConclusionThe combination of MSC exosomes and fibrin sealant (Exosome+Fibrin) applied to a suture repair enhanced the morphologic and histologic properties of the ACL in a rabbit model, and these improvements could be attributed to the augmented functions of ACL fibroblasts with exosome treatment.Clinical relevanceThis work supports the use of MSC exosomes in biological augmentation of ACL healing after suture repair.

Project description:BackgroundCell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing.MethodsMesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics.ResultsPHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05).ConclusionsSilencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

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Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

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Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

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