MSC exosomes mediate cartilage repair by enhancing proliferation, attenuating apoptosis and modulating immune reactivity

MSC exosomes alleviate temporomandibular joint osteoarthritis by attenuating inflammation and restoring matrix homeostasis.

Cues from the extracellular environment, including physical stimuli, are well known to affect mesenchymal stem cell (MSC) properties in terms of proliferation and differentiation. Many therapeutic strategies are now targeting this knowledge to increase the efficacy of cell therapies, typically employed to repair tissue functions in the event of injury, either by direct engraftment into the target tissue or differentiation into mature tissues. However, it is now envisioned that harnessing the repertoire of factors secreted by MSCs (termed the secretome) may provide an alternate to these cell therapies. Of current interest are both direct protein secretions and two major subpopulations of bioactive extracellular vesicles (EVs), namely exosomes and microvesicles. EVs released by MSCs are reflective of their cells of origin, able to impact upon the activities of other cells in the local microenvironment, making the rational design of MSC paracrine activities an encouraging strategy to reproducibly modulate cell therapies. The precise mechanisms by which the secretome is modulated by the microenvironment, however, remain elusive. Controlling MSC growth conditions with oxygen tension, growth factor composition, and mechanical properties may serve to directly influence paracrine activity. Our growing understanding implicates components of the mechanotransduction machinery in translating both mechanical and chemical cues from the environment into alterations in gene regulation and varied paracrine activity. As technologies are developed to manufacture MSCs, advances in bioengineering and novel insight of how the extracellular environment affects MSC paracrine activity will play a pivotal role in the generation of widespread, successful, clinical MSC therapies.

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Effect of the Microenvironment on Mesenchymal Stem Cell Paracrine Signaling: Opportunities to Engineer the Therapeutic Effect

Exosomes, nanoscale extracellular vesicles functioning as cell-to-cell communicators, are an emerging promising therapeutic in the field of bone tissue engineering. Here, we report the construction and evaluation of a novel cell-free tissue-engineered bone that successfully accelerated the restoration of critical-sized mouse calvarial defects through combining exosomes derived from human adipose-derived stem cells (hASCs) with poly(lactic-co-glycolic acid) (PLGA) scaffolds. The exosomes were immobilized on the polydopamine-coating PLGA (PLGA/pDA) scaffolds under mild chemical conditions. Specifically, we investigated the effects of hASC-derived exosomes on the osteogenic, proliferation, and migration capabilities of human bone marrow-derived mesenchymal stem cells in vitro and optimized their osteoinductive effects through osteogenic induction. Furthermore, an in vitro assay showed exosomes could release from PLGA/pDA scaffold slowly and consistently and in vivo results showed this cell-free system enhanced bone regeneration significantly, at least partially through its osteoinductive effects and capacities of promoting mesenchymal stem cells migration and homing in the newly formed bone tissue. Therefore, overall results demonstrated that our novel cell-free system comprised of hASC-derived exosomes and PLGA/pDA scaffold provides a new therapeutic paradigm for bone tissue engineering and showed promising potential in repairing bone defects.

Tissue-Engineered Bone Immobilized with Human Adipose Stem Cells-Derived Exosomes Promotes Bone Regeneration

Osteoarthritis (OA) is the most common joint disease worldwide. In the past decade, mesenchymal stem cells (MSCs) have been used widely for the treatment of OA. A potential mechanism of MSC-based therapies has been attributed to the paracrine secretion of trophic factors, in which exosomes may play a major role. In this study, we aimed to compare the effectiveness of exosomes secreted by synovial membrane MSCs (SMMSC-Exos) and exosomes secreted by induced pluripotent stem cell-derived MSCs (iMSC-Exos) on the treatment of OA. Induced pluripotent stem cell-derived MSCs and synovial membrane MSCs were characterized by flow cytometry. iMSC-Exos and SMMSC-Exos were isolated using an ultrafiltration method. Tunable resistive pulse-sensing analysis, transmission electron microscopy, and western blots were used to identify exosomes. iMSC-Exos and SMMSC-Exos were injected intra-articularly in a mouse model of collagenase-induced OA and the efficacy of exosome injections was assessed by macroscopic, histological, and immunohistochemistry analysis. We also evaluated the effects of iMSC-Exos and SMMSC-Exos on proliferation and migration of human chondrocytes by cell-counting and scratch assays, respectively. The majority of iMSC-Exos and SMMSC-Exos were approximately 50–150 nm in diameter and expressed CD9, CD63, and TSG101. The injection of iMSC-Exos and SMMSC-Exos both attenuated OA in the mouse OA model, but iMSC-Exos had a superior therapeutic effect compared with SMMSC-Exos. Similarly, chondrocyte migration and proliferation were stimulated by both iMSC-Exos and SMMSC-Exos, with iMSC-Exos exerting a stronger effect. The present study demonstrated that iMSC-Exos have a greater therapeutic effect on OA than SMMSC-Exos. Because autologous iMSCs are theoretically inexhaustible, iMSC-Exos may represent a novel therapeutic approach for the treatment of OA.

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Comparison of exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells and synovial membrane-derived mesenchymal stem cells for the treatment of osteoarthritis

Recently, accumulating evidence has shown that exosomes, the naturally secreted nanocarriers of cells, can exert therapeutic effects in various disease models in the absence of parent cells. However, application of exosomes in bone defect repair and regeneration has been rarely reported, and little is known regarding their underlying mechanisms. Exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects, and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation, migration, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting, scratch assays, and qRT-PCR, respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair. We found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition, the internalization of exosomes into hBMSCs could profoundly enhance the proliferation, migration, and osteogenic differentiation of hBMSCs. Furthermore, gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs. We propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs, which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These results indicate that naturally secreted nanocarriers-exosomes can be used as a bioactive material to improve the bioactivity of the biomaterials, and that hiPS-MSC-Exos combined with β-TCP scaffolds can be potentially used for repairing bone defects.

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Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway.

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1α (HIF-1α) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1α in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study, we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1α and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1α. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats, DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone, leading to bone regeneration. Collectively, the results indicate that DMOG, via activation of the PI3K/Akt pathway, promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration.

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Dimethyloxaloylglycine Promotes the Angiogenic Activity of Mesenchymal Stem Cells Derived from iPSCs via Activation of the PI3K/Akt Pathway for Bone Regeneration.

Neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) are becoming an appealing source of cell-based therapies of brain diseases. As such, it is important to understand the molecular mechanisms that regulate the differentiation of iPSCs toward NSCs. It is well known that Notch signaling governs the retention of stem cell features and drives stem cells fate. However, further studies are required to investigate the role of Notch signaling in the NSCs differentiation of iPSCs. In this study, we successfully generated NSCs from human iPSCs using serum-free medium supplemented with retinoic acid (RA) in vitro. We then assessed changes in the expression of Notch signaling-related molecules and some miRNAs (9, 34a, 200b), which exert their regulation by targeting Notch signaling. Moreover, we used a γ-secretase inhibitor (DAPT) to disturb Notch signaling. Data revealed that the levels of the Notch signaling-related molecules decreased, whereas those miRNAs increased, during this differentiation process. Inhibition of Notch signaling accelerated the formation of the neural rosette structures and the expression of NSC and mature neurocyte marker genes. This suggests that Notch signaling negatively regulated the neuralization of human iPSCs, and that this process may be regulated by some miRNAs.

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Chunyuan Chen

Papers

Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway.

Dimethyloxaloylglycine Promotes the Angiogenic Activity of Mesenchymal Stem Cells Derived from iPSCs via Activation of the PI3K/Akt Pathway for Bone Regeneration.

Inhibition of Notch signaling facilitates the differentiation of human-induced pluripotent stem cells into neural stem cells