Chronic respiratory diseases are diseases of the airways and other structures of the lung, mainly including chronic obstructive pulmonary disease (COPD), asthma, occupational lung diseases and pulmonary hypertension (World Health Organization, 2021). Chronic respiratory diseases remain leading causes of death and disability. It is estimated that 4 million people per year die from chronic respiratory diseases worldwide (Forum of International Respiratory Societies, 2017; Soriano et al., 2020). Considering the heavy social burden from chronic respiratory diseases, effective prevention and treatment measures are urgently needed.

Mesenchymal stem cells (MSCs) refer to a group of cells from bone marrow, fat and umbilical cord, which have the ability of adherent growth. Currently, MSCs are commonly used in cell therapy due to important potentials of proliferation, differentiation and immune regulation (Weiss and Dahlke, 2019). Extracellular vesicles (EVs) are vesicles with membrane structure released by cells in the state of activation, injury or apoptosis; According to the diameter of vesicles, they can be divided into apoptotic bodies, micro vesicles, and exosomes (Shao et al., 2018). A growing number of studies have shown that EVs mediate intercellular targeted regulation by transferring different substances (including protein, DNA, microRNA, lncRNA, circRNA and mRNA) (Lasda and Parker, 2016; Li et al., 2019; Vagner et al., 2019; Vu et al., 2020; Vilaca et al., 2021). Importantly, compared with other commonly used drug delivery carriers (such as liposomes), EVs have the advantages of high internal targeting ability, low immunogenicity, high modification flexibility and high biological barrier permeability, which opens up a new field for modern drug delivery (Herrmann et al., 2021).

MSCs can produce a large number of EVs. MSC-EVs can mediate the interaction between MSCs and target cells through delivering genetic and protein goods, and this targeting mode has attracted great attention in the treatment of chronic kidney diseases, cardiovascular diseases, neurological diseases and tumor (Eirin and Lerman, 19792021; Fu et al., 2020; Tran et al., 2020; Pucci et al., 2021)) MSC-EVs can play a therapeutic role similar to MSCs, and MSC-EVs are easier to store and make dosage forms; Meanwhile, MSC-EVs avoid some limitations of cell therapy, such as embolization and tumorigenesis (Shi et al., 2021). The potential anti-inflammatory effect of MSC-EVs has been proved in several previous studies related to acute lung injury (Monsel et al., 2015; Ahn et al., 2018; Khatri et al., 2018). Recently, more and more researches have focused on the therapeutic role of MSC-EVs in chronic respiratory diseases. In this review, we summarize the cumulative strategies and mechanisms of MSC-EVs in treating chronic respiratory diseases, which may provide novel therapeutic alternatives for chronic respiratory diseases.

Umbilical cord (UC) and bone marrow (BM) are main sources of MSC-EVs in treating chronic respiratory diseases, while other relatively rare sources include adipose tissue, lung, and induced pluripotent stem cells (iPSCs).

UC is an appropriate source of MSC-EVs, with the advantage of non-invasion in the collecting process of UC (de Faria et al., 2012). BM may be suboptimal, as BM-MSCs have shown decreased differentiation potential and BM samples are rather difficult in acquisition (Sergejeva et al., 2004). Of note, the use of adipose tissues to harvest MSCs has become common due to its unique advantages such as easy access, great texture and rapid expansion in vitro (Bertassoli et al., 2013; Jalali and Rabiee, 2016). However, few studies have investigated the therapeutic functions of MSC-EVs from adipose tissues in chronic respiratory diseases. Lung is another source of MSC-EVs. Cells derived from human lung allografts present a multipotent mesenchymal cell population, which is locally resident in the human adult lung and has extended life span in vivo; And multipotency of these cells was proved by their capacity to differentiate into adipocytes, chondrocytes, and osteocytes (Lama et al., 2007). iPSCs are also sources of MSC-EVs, and MSC-EVs from iPSCs can be utilized for drug delivery by loading with proteins (Yuan et al., 2017). iPSCs maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers, which provides a novel direction of regenerative medicine.

Methods used to isolate MSC-EVs in treating chronic respiratory diseases involve ultracentrifugation (UCF), exosome extraction kits and anion-exchange chromatography.

UCF is the current gold standard and the most studied methods for EV isolation (Momen-Heravi, 2017). The centrifugal speed for microvesicles is 10,000–60,000 g, while 100,000–200,000 g for exosomes (Borges et al., 2013). However, UCF is time-consuming and isolation outcomes are also highly dependent on speed, rotor type as well as temperature (Livshits et al., 2015). Also, UCF could lead to aggregated EVs after pelleting (Linares et al., 2015). Exosome extraction kits are emerging tools for exosome isolation. One previous study demonstrated that ultracentrifugation and exosome extraction kits are two complementary approaches allowing the detection of different proteoforms with various abundance and purity levels (Gemoll et al., 2020). Thereby, combined isolation methods may be optional to consider in future studies. Anion-exchange chromatography is a scalable protocol developed by Kim et al., in 2016 (Kim et al., 2016). They found that most of the protein from EVs in the harvested medium was bound to an anion exchange resin but that little bound to a cation exchange resin. Besides above mentioned methods, additional techniques or combinations of techniques have been developed to isolate EVs including field-flow fractionation, variations on size exclusion chromatography, microfiltration, asymmetric flow field-flow fractionation, field-free viscoelastic flow, tangential flow filtration and variations thereon, alternating current electrophoretics, deterministic lateral displacement arrays, fluorescence-activated sorting, novel immunoisolation or other affinity isolation technologies etc. (Théry et al., 2018)

The dose of MSC-EVs varies in different disease models. Most studies measured total protein amount using various colorimetric assays to indirectly suggest the particle number of EVs. In vivo, 15–100 μg (protein) is the commonly used EV dose for treating mouse or rat models; while 2–40 μg/ml is the usual treatment dose for studies in vitro. Some studies used absolute particle count or MSC number to measure EV treatment dose. The absolute particle count of EVs ranges from 2×10⁷ to 2×10¹⁰ in vivo.

Intratracheal delivery and intravenous administration are main routes of MSC-EVs in the treatment of chronic respiratory diseases. Compared with intravenous administration, intratracheal administration is a more direct and accurate treatment mode (Chen et al., 2021). An intraperitoneal route could be selected based on its advantages for multiple or daily treatments of EVs (Maremanda et al., 2019). Details of therapeutic strategies of MSC-EVs in treating chronic respiratory diseases are demonstrated in Table 1.

Collectively, emerging findings reveal that MSC-EVs may serve as a novel cell-free-based therapy for chronic respiratory diseases, including COPD, asthma, pulmonary fibrosis, and pulmonary arterial hypertension. Bone marrow and umbilical cord are main sources of MSC-EVs in treating chronic respiratory diseases, while adipose tissue, lung, and induced pluripotent stem cells are also applied. Isolation methods of MSC-EVs in treating chronic respiratory diseases involve ultracentrifugation, exosome extraction kits and anion-exchange chromatography. As for treatment routes of MSC-EVs, intratracheal delivery and intravenous administration are most widely used. Current studies suggest that MSC-EVs are able to transfer microRNAs and protein to target cells and further magnify the therapeutic effects. However, existing studies have not compared treatment effects among MSC-EVs from different sources (umbilical cord, bone marrow, adipose tissue and induced pluripotent stem cells) or types (apoptotic bodies, microvesicles and exosomes), which highly prompt future studies to address this item. Novel administration routes of MSC-EVs, such as nebulized inhalation, are also worthy of attention so as to improve the safety and effectiveness of MSC-EVs in the treatment of chronic respiratory diseases. Moreover, future studies through nucleic acid sequencing and proteomics are still warranted to reveal internal components of MSC-EVs from various sources and explicit mechanisms of MSC-EVs treatment on different chronic respiratory disease models.