Sally Yunsun Kim

Many age-related lung diseases encompass aberrant repair mechanisms. To develop novel treatments for these diseases, it is critical to understand how biology and repair signals are altered in aged lungs. Extracellular vesicles (EVs) carry bioactive signals reflecting their source tissue; therefore, we used EVs derived from precision-cut lung slices (PCLS) as tools to investigate differences in lung biology and repair signals that occur upon ageing and injury. We compared the physicochemical properties and biological content of four groups of EVs, obtained from uninjured and injured, young and aged PCLS. Treatment with EVs obtained from young, uninjured (YU) PCLS decreased apoptotic cells in both young and aged spatially injured (AIR)-PCLS. YU EVs also increased the percentage of alveolar progenitor cells in aged AIR-PCLS, indicating that aged lungs retain the capacity to mount a repair response, if provided with the right biological cues. Small RNA sequencing revealed the miRNA content of each EV group is altered, depending on the physiological conditions of their source tissue. Bioinformatic analysis of the target genes regulated by differentially expressed miRNAs identified enrichment of genes required for lung development and repair. These findings pave the way for future therapeutic strategies to repair or regenerate aged lungs.

Aging is a complex feature and involves loss of multiple functions and nonreversible phenotypes. However, several studies suggest it is possible to protect against aging and promote rejuvenation. Aging is associated with many factors, such as telomere shortening, DNA damage, mitochondrial dysfunction, and loss of homeostasis. The integrity of the cytoskeleton is associated with several cellular functions, such as migration, proliferation, degeneration, and mitochondrial bioenergy production, and chronic disorders, including neuronal degeneration and premature aging. Cytoskeletal integrity is closely related with several functional activities of cells, such as aging, proliferation, degeneration, and mitochondrial bioenergy production. Therefore, regulation of cytoskeletal integrity may be useful to elicit antiaging effects and to treat degenerative diseases, such as dementia. The actin cytoskeleton is dynamic because its assembly and disassembly change depending on the cellular status. Aged cells exhibit loss of cytoskeletal stability and decline in functional activities linked to longevity. Several studies reported that improvement of cytoskeletal stability can recover functional activities. In particular, microtubule stabilizers can be used to treat dementia. Furthermore, studies of the quality of aged oocytes and embryos revealed a relationship between cytoskeletal integrity and mitochondrial activity. This review summarizes the links of cytoskeletal properties with aging and degenerative diseases and how cytoskeletal integrity can be modulated to elicit antiaging and therapeutic effects.

Current medicine has only taken us so far in reducing disease and tissue damage. Extracellular vesicles (EVs), which are membranous nanostructures produced naturally by cells, have been hailed as a next-generation medicine. EVs deliver various biomolecules, including proteins, lipids and nucleic acids, which can influence the behaviour of specific target cells. Since EVs not only mirror composition of their parent cells but also modify the recipient cells, they can be used in three key areas of medicine: regenerative medicine, disease detection and drug delivery. In this Review, we discuss the transformational and translational progress witnessed in EV-based medicine to date, focusing on two key elements: the mechanisms by which EVs aid tissue repair (for example, skin and bone tissue regeneration) and the potential of EVs to detect diseases at an early stage with high sensitivity and specificity (for example, detection of glioblastoma). Furthermore, we describe the progress and results of clinical trials of EVs and demonstrate the benefits of EVs when compared with traditional medicine, including cell therapy in regenerative medicine and solid biopsy in disease detection. Finally, we present the challenges, opportunities and regulatory framework confronting the clinical application of EV-based products.

Recent advances in cell culture models like air‒liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three‐dimensional environment of the lung. Precision‐cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR‐PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi‐high‐throughput screening of potential therapeutics. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Generation of precision‐cut lung slicesBasic Protocol 2: The acid injury and repair modelBasic Protocol 3: Analysis of AIR‐PCLS: Immunostaining and imaging

A number of medicines are currently under investigation for the treatment of COVID-19 disease including anti-viral, anti-malarial, and anti-inflammatory agents. While these treatments can improve patient's recovery and survival, these therapeutic strategies do not lead to unequivocal restoration of the lung damage inflicted by this disease. Stem cell therapies and, more recently, their secreted extracellular vesicles (EVs), are emerging as new promising treatments, which could attenuate inflammation but also regenerate the lung damage caused by COVID-19. Stem cells exert their immunomodulatory, anti-oxidant, and reparative therapeutic effects likely through their EVs, and therefore, could be beneficial, alone or in combination with other therapeutic agents, in people with COVID-19. In this review article, we outline the mechanisms of cytokine storm and lung damage caused by SARS-CoV-2 virus leading to COVID-19 disease and how mesenchymal stem cells (MSCs) and their secreted EVs can be utilized to tackle this damage by harnessing their regenerative properties, which gives them potential enhanced clinical utility compared to other investigated pharmacological treatments. There are currently 17 clinical trials evaluating the therapeutic potential of MSCs for the treatment of COVID-19, the majority of which are administered intravenously with only one clinical trial testing MSC-derived exosomes via inhalation route. While we wait for the outcomes from these trials to be reported, here we emphasize opportunities and risks associated with these therapies, as well as delineate the major roadblocks to progressing these promising curative therapies toward mainstream treatment for COVID-19.

Degenerative lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis are incurable. The genesis of these diseases is either an inadequate or aberrant tissue repair processes yet current treatments cannot actively repair injured tissue. A promising approach to drive tissue repair is the stimulation of Wnt signalling, which has a crucial role in lung epithelial development and alveolar epithelial cell function in lung disease (Uhl FE, et al. Eur Respir J. 2015, 46(4):1150). However, the precise molecular mechanisms that target the pathway to drive repair are not well established. Pharmacological modification of the Wnt pathway is a promising approach to stimulate lung repair. To investigate the efficacy of novel pro-repair molecules, an acid drop injury is made on a spatially restricted area of mouse precision cut lung slices (PCLS). Key aspects of tissue repair are assessed at 24, 48, 72 h by automated quantification of cell proliferation and differentiation (Ki67 and Hopx) and pro-surfactant protein C (proSp-C, a marker for ATII cells/an early repair response, Fig. 1A). Post imaging, additional analysis is conducted on injured and non-injured areas of PCLS (Fig. 1B) by qPCR to analyse pro-repair genes (Fig. 1C). This study reports the establishment of a novel ex-vivo model for studies of lung injury and repair. The techniques developed in this work are likely to provide a versatile new tool in respiratory research.

Many acute and chronic lung injuries are incurable and rank as the fourth leading cause of death globally. While stem cell treatment for lung injuries is a promising approach, there is growing evidence that the therapeutic efficacy of stem cells originates from secreted extracellular vesicles (EVs). Consequently, EVs are emerging as next‐generation therapeutics. While EVs are extensively researched for diagnostic applications, their therapeutic potential to promote tissue repair is not fully elucidated. By housing and delivering tissue‐repairing cargo, EVs refine the cellular microenvironment, modulate inflammation, and ultimately repair injury. Here, the potential use of EVs derived from two placental mesenchymal stem/stromal cell (MSC) lines is presented; a chorionic MSC line (CMSC29) and a decidual MSC cell line (DMSC23) for applications in lung diseases. Functional analyses using in vitro models of injury demonstrate that these EVs have a role in ameliorating injuries caused to lung cells. It is also shown that EVs promote repair of lung epithelial cells. This study is fundamental to advancing the field of EVs and to unlock the full potential of EVs in regenerative medicine.