Mechanisms underlying the protective effects of mesenchymal stem cell-based therapy.

Mesenchymal stem cells (MSCs) have been extensively investigated for the treatment of various diseases. The therapeutic potential of MSCs is attributed to complex cellular and molecular mechanisms of action including differentiation into multiple cell lineages and regulation of immune responses via immunomodulation. The plasticity of MSCs in immunomodulation allow these cells to exert different immune effects depending on different diseases. Understanding the biology of MSCs and their role in treatment is critical to determine their potential for various therapeutic applications and for the development of MSC-based regenerative medicine. This review summarizes the recent progress of particular mechanisms underlying the tissue regenerative properties and immunomodulatory effects of MSCs. We focused on discussing the functional roles of paracrine activities, direct cell-cell contact, mitochondrial transfer, and extracellular vesicles related to MSC-mediated effects on immune cell responses, cell survival, and regeneration. This will provide an overview of the current research on the rapid development of MSC-based therapies.

Increased Group 2 Innate Lymphoid Cells Are Correlated with Eosinophilic Granulocytes in Patients with Allergic Airway Inflammation.

The T helper 2 (Th2)-type response was considered the hypostasis of allergic airway diseases, including asthma and allergic rhinitis (AR). However, more recent studies have suggested that allergic airway inflammation also depends on innate immunity and is closely related to group 2 innate lymphoid cells (ILC2s). This study evaluated the ILC2 levels of asthma subjects, patients with asthma and AR, and healthy individuals, regarding how to investigate the relationship between clinical data and ILC2 levels. It was found that asthma patients and asthma with AR patients had higher ILC2 levels compared to healthy subjects. ILC2s were positively correlated with the percentage of eosinophils in patients with asthma and AR, but not with pulmonary function. ILC2 levels were higher in mild asthma subjects than in patients with severe asthma. This study provides a new interpretation of the pathogenesis of allergic airway inflammation and may provide a new direction for the diagnosis and assessment of allergic airway diseases.

Increased innate type 2 immune response in house dust mite-allergic patients with allergic rhinitis.

Group 2 innate lymphoid cells (ILC2s) are essential in initiating and driving allergic immune responses. However, there were inconsistent findings of the ILC2 levels in allergic rhinitis (AR) patients. This study investigated the ILC2 levels in the peripheral blood of house dust mite (HDM)-sensitized AR patients and their ability to secrete type 2 cytokines. The levels of ILC2s with phenotypic ILC2 characteristics were increased in the HDM-AR patients. The AR patients' symptom score and IL-13 levels were positively associated with the ILC2s in HDM-AR patients. The epithelial cytokine stimulation induced dramatic production of IL-5 and IL-13 in PBMCs of AR patients. We successfully sorted ILC2s from AR patients and identified their ability of type 2 cytokines production. The number of ILC2s increased in the HDM-AR patients and ILC2s produced the amount of TH2 cytokines in the presence of epithelial cytokines, which suggested the important role of ILC2 in AR patients.

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells.

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.

Small extracellular vesicles derived from human MSCs prevent allergic airway inflammation via immunomodulation on pulmonary macrophages.

Allergic airway inflammation is a major public health disease that affects up to 300 million people in the world. However, its management remains largely unsatisfactory. The dysfunction of pulmonary macrophages contributes greatly to the development of allergic airway inflammation. It has been reported that small extracellular vesicles derived from mesenchymal stromal cells (MSC-sEV) were able to display extensive therapeutic effects in some immune diseases. This study aimed to investigate the effects of MSC-sEV on allergic airway inflammation, and the role of macrophages involved in it. We successfully isolated MSC-sEV by using anion exchange chromatography, which were morphologically intact and positive for the specific EV markers. MSC-sEV significantly reduced infiltration of inflammatory cells and number of epithelial goblet cells in lung tissues of mice with allergic airway inflammation. Levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid were also significantly decreased. Importantly, levels of monocytes-derived alveolar macrophages and M2 macrophages were significantly reduced by MSC-sEV. MSC-sEV were excreted through spleen and liver at 24 h post-administration in mice, and were able to be taken in by macrophages both in vivo and in vitro. In addition, proteomics analysis of MSC-sEV revealed that the indicated three types of MSC-sEV contained different quantities of proteins and shared 312 common proteins, which may be involved in the therapeutic effects of MSC-sEV. In total, our study demonstrated that MSC-sEV isolated by anion exchange chromatography were able to ameliorate Th2-dominant allergic airway inflammation through immunoregulation on pulmonary macrophages, suggesting that MSC-sEV were promising alternative therapy for allergic airway inflammation in the future.

Small extracellular vesicles derived from human mesenchymal stromal cells prevent group 2 innate lymphoid cell-dominant allergic airway inflammation through delivery of miR-146a-5p.

Group 2 innate lymphoid cells (ILC2s) are recently reported to play a more critical role in allergic diseases. We previously identified that mesenchymal stromal cells (MSCs) elicited therapeutic effects on allergic airway inflammation. Small extracellular vesicles (sEV) derived from MSCs possess striking advantages including low immunogenicity and high biosafety, and is extremely promising cell-free therapeutic agents. However, the effects of MSC-sEV on ILC2s are still unclear. Additionally, scalable isolation protocols are required for the mass production of homogenous MSC-sEV especially in clinical application. We previously reported that induced pluripotent stem cells-derived MSCs were the ideal cellular source for the large preparation of MSC-sEV. Here we developed a standardized scalable protocol of anion-exchange chromatography for isolation of MSC-sEV, and investigated the effects of MSC-sEV on ILC2 function from patients with allergic rhinitis and in a mouse ILC2-dominant asthma model. The characterization of MSC-sEV was successfully demonstrated in terms of size, morphology and specific markers. Using flow cytometry and human Cytokine Antibody Array, MSC-sEV but not fibroblasts-sEV (Fb-sEV) were found to significantly inhibit the function of human ILC2s. Similarly, systemic administration of MSC-sEV but not Fb-sEV exhibited an inhibition of ILC2 levels, inflammatory cell infiltration and mucus production in the lung, a reduction in levels of T helper 2 cytokines, and alleviation of airway hyperresponsiveness in a mouse model of asthma. Using RNA sequencing, miR-146a-5p was selected as the candidate to mediate the above effects of MSC-sEV. We next revealed the uptake of ILC2s to MSC-sEV, and that transfer of miR-146a-5p in MSC-sEV to ILC2s in part contributed to the effects of MSC-sEV on ILC2s in vitro and in a mouse model. In conclusion, we demonstrated that MSC-sEV were able to prevent ILC2-dominant allergic airway inflammation at least partially through miR-146a-5p, suggesting that MSC-sEV could be a novel cell-free strategy for the treatment of allergic diseases.

Mesenchymal stem cells for inflammatory airway disorders: promises and challenges.

The regenerative and immunomodulatory characteristics of mesenchymal stem cells (MSCs) make them attractive in the treatment of many diseases. Although they have shown promising preclinical studies of immunomodulation and paracrine effects in inflammatory airway disorders and other lung diseases, there are still challenges that have to be overcome before MSCs can be safely, effectively, and routinely applied in the clinical setting. A good understanding of the roles and mechanisms of the MSC immunomodulatory effects will benefit the application of MSC-based clinical therapy. In this review, we summarize the promises and challenges of the preclinical and clinical trials of MSC therapies, aiming to better understand the role that MSCs play in attempt to treat inflammatory airway disorders.

Human pluripotent stem cell-derived mesenchymal stem cells prevent chronic allergic airway inflammation via TGF-ß1-Smad2/Smad3 signaling pathway in mice.

BACKGROUND: Asthma is a chronic disease involving inflamed airways, which were previously demonstrated, can be modulated by the mesenchymal stem cells derived from induced pluripotent stem cells (iPSC-MSCs). However, the long-term effects of iPSC-MSCs in inflamed airways are still unidentified. This study investigated the long-term effects and potential mechanisms involved in the immunomodulatory effects of iPSC-MSCs in the chronic mouse asthma model. METHODS: Both human iPSC-MSCs and bone marrow (BM)-MSCs were transplanted into the long-term ovalbumin-induced mice before sensitization phase or during the challenge phase. Airway hyper-respnsiveness measurement, immunohistochemistry and ELISA were employed to assess the effects of MSCs. In addition, Smad2/3 levels were assessed by western blot analysis to investigate the possible mechanism involved. RESULTS: The systemic administration of human iPSC-MSCs before the challenge protected the mice from the characters of the chronic allergic airway inflammation, in particular improving the airway remodeling and preventing fibrosis. In addition, the TGF-ß1/Smad pathway was identified involved in the immunomodulatory effects of iPSC-MSCs on chronic allergic airway inflammation. CONCLUSIONS: The study demonstrated that iPSC-MSCs are capable of preventing chronic allergic airway inflammation over a prolonged period, which further proved the iPSC-MSC therapeutic potential for allergic airway inflammation in a clinical scenario.

An in Vitro and in Vivo Study of the Effect of Dexamethasone on Immunoinhibitory Function of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells.

Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) represent a promising cell source for patient-specific cell therapy. We previously demonstrated that they display an immunomodulatory effect on allergic airway inflammation. Glucocorticoids are powerful anti-inflammatory compounds and widely used in the therapy of allergic diseases. However, the effect of glucocorticoids on the immunomodulatory function of iPSC-MSCs remains unknown. This study aimed to determine the effect of dexamethasone (Dex) on the immunomodulatory function of iPSC-MSCs in vitro and in vivo. A total of three human iPSC-MSC clones were generated from amniocyte-derived iPSCs. Anti-CD3/CD28-induced peripheral blood mononuclear cell (PBMC) proliferation was used to assess the effect of Dex on the immunoinhibitory function of iPSC-MSCs in vitro. Mouse models of contact hypersensitivity (CHS) and allergic airway inflammation were induced, and the levels of inflammation in mice were analyzed with the treatments of iPSC-MSCs and Dex, alone and combined. The results showed that Dex did not interfere with the immunoinhibitory effect of iPSC-MSCs on PBMC proliferation. In CHS mice, simultaneous treatment with Dex did not affect the effect of iPSC-MSCs on the inflammation, both in regional draining lymph nodes and in inflamed ear tissue. In addition, co-administration of iPSC-MSCs with Dex decreased the local expression of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the ears of CHS mice. In the mouse model of allergic airway inflammation, iPSC-MSC treatment combined with Dex resulted in a similar extent of reduction in pulmonary inflammation as iPSC-MSCs or Dex treatment alone. In conclusion, Dex does not significantly affect the immunomodulatory function of iPSC-MSCs both in vitro and in vivo. These findings may have implications when iPSC-MSCs and glucocorticoids are co-administered.

Connexin 43-Mediated Mitochondrial Transfer of iPSC-MSCs Alleviates Asthma Inflammation.

We previously identified an immunomodulatory role of human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSCs) in asthmatic inflammation. Mitochondrial transfer from bone marrow MSCs to epithelial cells can result in the attenuation of acute lung injury in mice. However, the effects of mitochondrial transfer from iPSC-MSCs to epithelial cells in asthma and the mechanisms underlying these effects are unclear. We found that iPSC-MSC transplantation significantly reduced T helper 2 cytokines, attenuated the mitochondrial dysfunction of epithelial cells, and alleviated asthma inflammation in mice. Tunneling nanotubes (TNTs) were formed between iPSC-MSCs and epithelial cells, and mitochondrial transfer from iPSC-MSCs to epithelial cells via TNTs was observed both in vitro and in mice. Overexpression or silencing of connexin 43 (CX43) in iPSC-MSCs demonstrated that CX43 plays a critical role in the regulation of TNT formation by mediating mitochondrial transfer between iPSC-MSCs and epithelial cells. This study provides a therapeutic strategy for targeting asthma inflammation.

The genes involved in asthma with the treatment of human embryonic stem cell-derived mesenchymal stem cells.

BACKGROUND: Asthma is affecting more than 300 million people worldwide, which represents the most common chronic disease among children. We previously found that mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) modulated the immune response on Th2-mediated asthma in vivo and in vitro. This study further evaluated the immunomodulatory effects of MSCs from human embryonic stem cells (hESCs) on asthma. METHODS: Multipotent hESC-MSCs were obtained using a feeder-free method. The hESC-MSCs were analysed for the expression of stem cell surface markers by flow cytometry, their differentiation potentials were analysed using in vitro trilineage differentiation methods hESC-MSCs were transplanted into the murine model with ovalbumin (OVA)-induced airway allergic inflammation. The expression levels of allergic related genes were measured by the mRNA PCR arrays. RESULTS: The hESC-MSCs expressed classical MSC markers and held the capability of differentiation into multiple mesoderm-type cell lineages. hESC-MSCs were able to suppress allergic inflammation by modulating Th2 cells and eosinophils in the mice, and reversed the reduction of regulatory T cells. By using PCR array, 5 mRNAs- chemokine (C-C motif) ligand 11 (Ccl11), Ccl24, interleukin13 (Il13), Il33 and eosinophil-associated, ribonuclease A family, member 11 (Ear11) were identified the most relevant in murine airway allergic inflammation and hESC-MSCs treatment. CONCLUSIONS: The therapeutic effects of hESC-MSCs were identified in the murine model of airway allergic inflammation with key mRNAs involved. This study will provide a better understanding regarding the mechanisms underlying hESC-MSCs therapeutic application in airway allergic inflammation.

Induced pluripotent stem cell-derived mesenchymal stem cells activate quiescent T cells and elevate regulatory T cell response via NF-κB in allergic rhinitis patients.

BACKGROUND: It has been demonstrated previously that induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSCs) have immunosuppressive effects on activated T cells. However, the effects of iPSC-MSCs on quiescent T cells are still unknown. The aim of this study was to identify the immunomodulatory role of iPSC-MSCs on resting peripheral blood mononuclear cells (PBMCs) from allergic rhinitis (AR) patients. METHODS: PBMCs were cocultured with iPSC-MSCs without any stimulation, following which lymphocyte proliferation, activation of T cells, TH1/TH2 and regulatory T (Treg) cell differentiation, and Treg cell function were analyzed. The roles of soluble factors and cell-cell contact were examined to investigate the mechanisms involved. RESULTS: iPSC-MSCs promoted the proliferation of resting lymphocytes, activated CD4+ and CD8+ T cells, and upregulated and activated Treg cells without any additional stimulation. In addition, iPSC-MSCs balanced biased TH1/TH2 cytokine levels. Cell-cell contact was confirmed to be a possible mechanism involved. NF-κB was identified to play an important role in the immunomodulatory effects of iPSC-MSCs on quiescent T cells. CONCLUSIONS: iPSC-MSCs activate quiescent T cells and elevate regulatory T-cell response in AR patients, suggesting different immunomodulatory functions of iPSC-MSCs according to the phases of diseases. Therefore, iPSC-MSCs are a potential therapeutic candidate for treating allergic airway inflammation.

MicroRNA-21 Mediates the Protective Effects of Mesenchymal Stem Cells Derived from iPSCs to Human Bronchial Epithelial Cell Injury Under Hypoxia.

Airway epithelial cell injury is a key triggering event to activate allergic airway inflammation, such as asthma. We previously reported that administration of mesenchymal stem cells (MSCs) significantly alleviated allergic inflammation in a mouse model of asthma, and the mmu-miR-21/ACVR2A axis may be involved. However, whether MSCs protect against bronchial epithelial cell injury induced by hypoxia, and the underlying mechanism, remain unknown. In our study, the human bronchial epithelial cell line BEAS-2B was induced to undergo apoptosis with a hypoxia mimic of cobalt chloride (CoCl2) damage. Treatment of MSCs derived from induced pluripotent stem cells (iPSCs) significantly decreased apoptosis of BEAS-2B cells. There was high miR-21 expression in injured BEAS-2B cells after MSC treatment. Transfection of the miR-21 mimic significantly decreased apoptosis of BEAS-2B, and transfection of a miR-21 inhibitor significantly increased apoptosis. More importantly, the protective effects of MSCs on injured BEAS-2B were reversed by transfection of the miR-21 inhibitor. Binding sites of human miR-21 were identified in the 3'UTR of human ACVR2A. We further determined that CoCl2 stimulation increased ACVR2A expression at both the mRNA and protein levels. Moreover, transfection of the miR-21 mimic further up-regulated ACVR2A expression induced by CoCl2, whereas transfection of the miR-21 inhibitor down-regulated ACVR2A expression. In addition, MSCs increased ACVR2A expression in BEAS-2B cells; however, this effect was reversed after transfection of the miR-21 inhibitor. Our data suggested that MSCs protect bronchial epithelial cells from hypoxic injury via miR-21, which may represent an important target. These findings suggest the potentially wide application of MSCs for epithelial cell injury during hypoxia.

Human iPSC-MSCs prevent steroid-resistant neutrophilic airway inflammation via modulating Th17 phenotypes.

BACKGROUND: Human induced pluripotent stem cells-derived mesenchymal stem cells (iPSC-MSCs) have been shown to be effective in Type 2 helper T cells (Th2)-dominant eosinophilic allergic airway inflammation. However, the role of iPSC-MSCs in Type 17 helper T cells (Th17)-dominant neutrophilic airway inflammation remains poorly studied. Therefore, this study was to explore the effects of iPSC-MSCs on an experimental mouse model of steroid-resistant neutrophilic airway inflammation and further determine the underlying mechanisms. METHODS: A mouse model of neutrophilic airway inflammation was established using ovalbumin (OVA) and lipopolysaccharide (LPS). Human iPSC-MSCs were systemically administered, and the lungs or bronchoalveolar lavage fluids (BALF) were collected at 4 h and 48 h post-challenge. The pathology and inflammatory cell infiltration, the T helper cells, T helper cells-associated cytokines, nuclear transcription factors and possible signaling pathways were evaluated. Human CD4+ T cells were polarized to T helper cells and the effects of iPSC-MSCs on the differentiation of T helper cells were determined. RESULTS: We successfully induced the mouse model of Th17 dominant neutrophilic airway inflammation. Human iPSC-MSCs but not dexamethasone significantly prevented the neutrophilic airway inflammation and decreased the levels of Th17 cells, IL-17A and p-STAT3. The mRNA levels of Gata3 and RORγt were also decreased with the treatment of iPSC-MSCs. We further confirmed the suppressive effects of iPSC-MSCs on the differentiation of human T helper cells. CONCLUSIONS: iPSC-MSCs showed therapeutic potentials in neutrophilic airway inflammation through the regulation on Th17 cells, suggesting that the iPSC-MSCs could be applied in the therapy for the asthma patients with steroid-resistant neutrophilic airway inflammation.

The lncRNAs involved in mouse airway allergic inflammation following induced pluripotent stem cell-mesenchymal stem cell treatment.

BACKGROUND: We have previously reported that induced pluripotent stem cell (iPSC)-mesenchymal stem cells (MSCs) alleviated asthma inflammation in mice. Long noncoding RNAs (lncRNAs) were recently reported as being involved in the immune responses. However, whether lncRNAs are associated with iPSC-MSC immunomodulation in allergic inflammation is still unclear. METHODS: Mice were induced into an asthmatic state and received treatment consisting of iPSC-MSCs. Memory T cells isolated from sensitized mice were challenged and co-cultured with iPSC-MSCs in vitro. Total RNA from the lungs and separated T cells were processed with an lncRNA/mRNA microarray. A series of bioinformatics technologies were used to screen the target lncRNAs. RESULTS: iPSC-MSCs significantly prevented asthma inflammation and decreased the Th2 cytokine levels. Over 1300 lncRNAs were differentially expressed after the induction of asthma, and 846 or 4176 lncRNAs were differentially expressed with iPSC-MSC treatment in mice or in vitro, respectively. After overlapping the differentially expressed lncRNAs produced in a similar manner in mice and in vitro, 23 lncRNAs and 96 mRNAs were selected, in which 58 protein-coding genes were predicted to be potential targets of the 23 lncRNAs. Furthermore, using a series of bioinformatics technologies, 9 lncRNAs co-expressed with the most differentially expressed mRNAs, which were enriched in terms of the immune response, were screened out via Pearson's correlation coefficient with mRNAs that were involved with inflammatory cytokines and receptors. lncRNAs MM9LINCRNAEXON12105+ and AK089315 were finally emphasized via quantitative real-time PCR validation. CONCLUSIONS: Our results suggested that aberrant lncRNA profiles were present after asthma induction and iPSC-MSC treatment, suggesting potential targets of allergic inflammation and iPSC-MSC-mediated immunomodulation.