cAMP Agonist Forskolin Disrupts Mitochondrial Metabolism and Induces Senescence in Human Mesenchymal Cells.

Adult-derived mesenchymal stem cells (MSCs) can be used in therapies for the treatment of various diseases. The MSCs derived from aging tissues or long-term MSC cultures could have diminished therapeutic effects compared with MSCs derived from younger tissues, but the underlying mechanism has not been completely established. Dysfunction of energy metabolism is one of the main mechanisms underlying cell senescence. Although cyclic adenosine monophosphate (cAMP) is known to inhibit cell division and proliferation in vitro, its impact on MSC senescence has not been described. In this study, we used forskolin, an adenylate cyclase agonist and cAMP inducer, to disrupt metabolism in human adipose-derived MSCs and investigate the effects of metabolic dysfunction on MSC senescence. Treatment of human MSCs with forskolin resulted in senescence phenotypes, including reduced proliferation, cell-cycle arrest, and enhanced expression of the cell aging markers p16 and p21. Further, the senescent MSCs exhibited increased adipogenesis capacity and decreased osteogenesis capacity as well as a senescence-associated secretory phenotype characterized by increased expression of several inflammatory factors. Forskolin-associated MSC senescence was mainly caused by oxidative stress-induced disruption of mitochondrial metabolism, and the senescent MSCs had high levels of reactive oxygen species and reduced sirtuin gene expression. Lastly, we found that cAMP inhibitor SQ22536 protects MSCs from forskolin-induced senescence and senescence-related inflammatory phenotype. Our results indicate that forskolin can cause senescence of human MSCs through oxidative stress-induced mitochondrial metabolic dysfunction, and thus the results provide a basis for developing strategies for improving the quality and efficacy of cultured MSCs for clinical use.

Short-Term Association of Air Pollutant Levels and Hospital Admissions for Stroke and Effect Modification by Apparent Temperature: Evidence From Shanghai, China.

The epidemiological evidence on relationships between air pollution, temperature, and stroke remains inconclusive. Limited evidence is available for the effect modification by apparent temperature, an indicator reflecting reactions to the thermal environment, on short-term associations between air pollution and hospital admissions for stroke. We used a generalized additive model with Poisson regression to estimate the relative risk (RR) of stroke admissions in Shanghai, China, between 2014 and 2016 associated with air pollutants, with subgroup analyses by age, sex, apparent temperature, and season. During the study period, changes in the daily number of stroke admissions per 10 µg/m3 increase in nitrogen dioxide (at lags 0, 1, 0-1, and 0-2) ranged from 1.05 (95% CI: 0.82%, 2.88%) to 2.24% (95% CI: 0.84%, 3.65%). For each 10 µg/m3 increase in sulfur dioxide concentrations at lags 1, 2, 0-1, and 0-2, the RR of daily stroke admissions increased by 3.34 (95% CI: 0.955%, 5.79%), 0.32 (95% CI: -1.97%, 2.67%), 3.33 (95% CI: 0.38%, 6.37%), and 2.86% (95% CI: -0.45%, 6.28%), respectively. The associations of same-day exposure to nitrogen dioxide with stroke admissions remained significant after adjustment for ozone levels. These associations were not modified by sex, age, apparent temperature, or season. More research is warranted to determine whether apparent temperature modifies the associations between air pollution and stroke admissions.

Mesenchymal stem cell treatment improves outcome of COVID-19 patients via multiple immunomodulatory mechanisms.

The infusion of coronavirus disease 2019 (COVID-19) patients with mesenchymal stem cells (MSCs) potentially improves clinical symptoms, but the underlying mechanism remains unclear. We conducted a randomized, single-blind, placebo-controlled (29 patients/group) phase II clinical trial to validate previous findings and explore the potential mechanisms. Patients treated with umbilical cord-derived MSCs exhibited a shorter hospital stay (P = 0.0198) and less time required for symptoms remission (P = 0.0194) than those who received placebo. Based on chest images, both severe and critical patients treated with MSCs showed improvement by day 7 (P = 0.0099) and day 21 (P = 0.0084). MSC-treated patients had fewer adverse events. MSC infusion reduced the levels of C-reactive protein, proinflammatory cytokines, and neutrophil extracellular traps (NETs) and promoted the maintenance of SARS-CoV-2-specific antibodies. To explore how MSCs modulate the immune system, we employed single-cell RNA sequencing analysis on peripheral blood. Our analysis identified a novel subpopulation of VNN2+ hematopoietic stem/progenitor-like (HSPC-like) cells expressing CSF3R and PTPRE that were mobilized following MSC infusion. Genes encoding chemotaxis factors - CX3CR1 and L-selectin - were upregulated in various immune cells. MSC treatment also regulated B cell subsets and increased the expression of costimulatory CD28 in T cells in vivo and in vitro. In addition, an in vivo mouse study confirmed that MSCs suppressed NET release and reduced venous thrombosis by upregulating kindlin-3 signaling. Together, our results underscore the role of MSCs in improving COVID-19 patient outcomes via maintenance of immune homeostasis.

Human Umbilical Cord Mesenchymal Stem Cells for Adjuvant Treatment of a Critically Ill COVID-19 Patient: A Case Report.

BACKGROUND: COVID-19 (coronavirus disease 2019) has become a global public health emergency since patients were first detected in Wuhan, China, in December 2019. Currently, there are no satisfying antiviral medications and vaccines available. CASE PRESENTATION: We reported the treatment process and clinical outcome of a 48-year-old man critically ill COVID-19 patient who received transfusion of allogenic human umbilical cord mesenchymal stem cells (UC-MSCs). CONCLUSIONS: We proposed that UC-MSC transfusion might be a new option for critically ill COVID-19. Although only one case we were shown, more similar clinical cases are inquired for further evidence providing the potential effectiveness of UC-MSC treatment.

Astragaloside IV Promotes Antiphotoaging by Enhancing the Proliferation and Paracrine Activity of Adipose-Derived Stem Cells.

Photoaging is a degenerative biological process. As a kind of pluripotent stem cells, adipose-derived stem cells (ADSCs) are widely used in the treatment of photoaging. Therefore, we aimed to find an effective way to improve the antiaging ability of ADSCs. In this study, we isolated ADSCs and assessed multilineage differentiation ability and markers. Cultured ADSCs were preconditioned with astragaloside IV (ASI) at 10-7, 10-6, and 10-5 M. Cell proliferation was assessed by CCK-8 assay and cytokine secretion by enzyme-linked immunosorbent assay (ELISA). A fibroblast photoaging model was established and cocultured with normal ADSCs or ASI-treated ADSCs. Matrix metalloproteinase-1 (MMP1) and type I procollagen (PC-I) secreted by human dermal fibroblasts were measured by ELISA. The effects of ASI-treated ADSCs on skin texture, including dermal thickness, collagen content, and microvessel density, in a photoaging animal model were analyzed using H&E staining, Masson staining, and CD31 immunohistochemistry, respectively. We found that 10-6 M ASI could significantly promote cell proliferation and stimulate robust secretion of growth factors in ADSCs. Furthermore, our data showed that ASI-treated ADSCs could markedly reverse the ultraviolet B-induced decrease of PC-I secretion and increase of MMP-1 release in fibroblasts. Moreover, in photoaged skin of nude mice, ASI-treated ADSCs significantly increased dermal thickness, collagen content, and microvessel density.

Transplantation of ACE2- Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia.

A coronavirus (HCoV-19) has caused the novel coronavirus disease (COVID-19) outbreak in Wuhan, China. Preventing and reversing the cytokine storm may be the key to save the patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs) have been shown to possess a comprehensive powerful immunomodulatory function. This study aims to investigate whether MSC transplantation improves the outcome of 7 enrolled patients with COVID-19 pneumonia in Beijing YouAn Hospital, China, from Jan 23, 2020 to Feb 16, 2020. The clinical outcomes, as well as changes of inflammatory and immune function levels and adverse effects of 7 enrolled patients were assessed for 14 days after MSC injection. MSCs could cure or significantly improve the functional outcomes of seven patients without observed adverse effects. The pulmonary function and symptoms of these seven patients were significantly improved in 2 days after MSC transplantation. Among them, two common and one severe patient were recovered and discharged in 10 days after treatment. After treatment, the peripheral lymphocytes were increased, the C-reactive protein decreased, and the overactivated cytokine-secreting immune cells CXCR3+CD4+ T cells, CXCR3+CD8+ T cells, and CXCR3+ NK cells disappeared in 3-6 days. In addition, a group of CD14+CD11c+CD11bmid regulatory DC cell population dramatically increased. Meanwhile, the level of TNF-α was significantly decreased, while IL-10 increased in MSC treatment group compared to the placebo control group. Furthermore, the gene expression profile showed MSCs were ACE2- and TMPRSS2- which indicated MSCs are free from COVID-19 infection. Thus, the intravenous transplantation of MSCs was safe and effective for treatment in patients with COVID-19 pneumonia, especially for the patients in critically severe condition.

Melatonin protects blood-brain barrier integrity and permeability by inhibiting matrix metalloproteinase-9 via the NOTCH3/NF-κB pathway.

The pathophysiological mechanism of white matter hyperintensities of cerebral small vessel disease (CSVD) includes an impaired blood-brain barrier (BBB) with increased permeability. Neuroinflammation likely contributes to the disruption of the BBB in CSVD. Therefore, understanding the molecular mechanism of how neuroinflammation causes BBB damage is essential to preventing BBB disruption in CSVD. Matrix metalloproteinase 9 (MMP-9) contributes to BBB damage in neuroinflammatory diseases. In this study, we observed that interleukin-1ß (IL-1ß)-induced MMP-9 secretion in pericytes increased BBB permeability to sodium fluorescein (Na-F) by damaging the disruption of VE-cadherin, occludin, claudin-5, and zonula occludin-1 (ZO-1). Melatonin reduced BBB permeability to Na-F and inhibited the disruption of the adherens and tight junction proteins. Melatonin also downregulated MMP-9 and upregulated tissue inhibitor of metalloproteinases 1 (TIMP-1) gene expression, which decreased the MMP-9/TIMP-1 ratio. In addition, nuclear translocation of NF-κB/p65 induced by IL-1ß in pericytes upregulated MMP-9 expression, which was inhibited by the NF-κB inhibitor PDTC. However, the NOTCH3 inhibitor DAPT significantly inhibited NF-κB/p65 translocation to the nucleus, while melatonin in combination with DAPT significantly prevented NF-κB/p65 translocation than DAPT alone. Our results suggest that melatonin reduced MMP-9-induced permeability of the BBB. Melatonin reduced MMP-9 expression and activity, which was induced by IL-1ß through the regulation of the NOTCH3/NF-κB signaling pathway in pericytes, suggesting that pericytes regulate BBB integrity and function.

Human Bone Marrow Mesenchymal Stem Cells Rescue Endothelial Cells Experiencing Chemotherapy Stress by Mitochondrial Transfer Via Tunneling Nanotubes.

Tunneling nanotubes (TNTs) are newly discovered tubular structures between two distant cells that facilitate the intercellular exchange of signals and components. Recent reports show that mesenchymal stem cells (MSCs) can rescue injured target cells and promote recovery from a variety of stresses via TNT-mediated mitochondrial transfer. In this study, we explored how TNTs form between bone marrow MSCs and endothelial cells (ECs) by using a human umbilical cord vein endothelial cell (HUVEC) model. TNT formation between MSCs and HUVECs could be induced by treating HUVECs with cytarabine (Ara-C), and human bone marrow mesenchymal stem cells (hBMMSCs) could transfer mitochondria to injured HUVECs through TNTs. Mitochondrial transfer from hBMMSCs to HUVECs via TNTs rescued the injured HUVECs by reducing apoptosis, promoting proliferation and restoring the transmembrane migration ability as well as the capillary angiogenic capacity of HUVECs. This study provides novel insights into the cell-cell communication between MSCs and ECs and supports the results of prior studies indicating that ECs promote hematopoietic regeneration. An improved understanding of MSC-EC cross-talk will promote the development of MSC-directed strategies for improving EC function and hematopoietic system regeneration following myelosuppressive and myeloablative injuries.

Mesenchymal stem cells and immune disorders: from basic science to clinical transition.

As a promising candidate seed cell type in regenerative medicine, mesenchymal stem cells (MSCs) have attracted considerable attention. The unique capacity of MSCs to exert a regulatory effect on immunity in an autologous/allergenic manner makes them an attractive therapeutic cell type for immune disorders. In this review, we discussed the current knowledge of and advances in MSCs, including its basic biological properties, i.e., multilineage differentiation, secretome, and immunomodulation. Specifically, on the basis of our previous work, we proposed three new concepts of MSCs, i.e., "subtotipotent stem cell" hypothesis, MSC system, and "Yin and Yang" balance of MSC regulation, which may bring new insights into our understanding of MSCs. Furthermore, we analyzed data from the Clinical Trials database ( http://clinicaltrials.gov ) on registered clinical trials using MSCs to treat a variety of immune diseases, such as graft-versus-host disease, systemic lupus erythematosus, and multiple sclerosis. In addition, we highlighted MSC clinical trials in China and discussed the challenges and future directions in the field of MSC clinical application.

Low level laser (LLL) attenuate LPS-induced inflammatory responses in mesenchymal stem cells via the suppression of NF-κB signaling pathway in vitro.

BACKGROUND: Considering promising results in animal models and patients, therapeutic use of MSCs for immune disease is likely to undergo continued evaluation. Low-lever laser (LLL) has been widely applied to retard the inflammatory reaction. LLL treatment can potentially be applied in anti-inflammatory therapy followed by stem cell therapy. AIM OF THE STUDY: The purpose of this study was to investigate the effect of LLL (660 nm) on the inflammatory reaction induced by LPS in human adipose derived mesenchymal stem cells (hADSCs) and pertinent mechanism. MATERIALS AND METHODS: Anti-inflammatory activity of LLL was investigated by LPS-induced mesenchymal stem cells. The production and expression of pro-inflammatory cytokines were evaluated by ELISA kits and RT-qPCR. Nuclear translocation of NF-κB was indicated by immunofluorescent staining. Phosphorylation status of NF-κB p65 and IκBα were illustrated by western blot assay. ROS generation was measured with CM-H2DCFDA, and NO secretion was determined by DAF-FM. We studied surface expression of lymphocyte activation markers when Purified peripheral blood mononuclear cell (PBMC) were activated by phytohaemagglutinin (PHA) in the presence of 3 types of treated MSCs. RESULTS: LLL reduced the secretion of IL-1ß, IL-6, IL8, ROS and NO in LPS treated MSCs. Immunofluorescent assay demonstrated the nuclear translocation decrease of NF-κB in LLL treated LPS induced MSCs. Western blot analysis also suggested that LLL suppressed NF-κB activation via regulating the phosphorylation of p65 and IκBα. MSC significantly reduced the expression of activation markers CD25 and CD69 on PHA-stimulated lymphocytes. CONCLUSION: The results indicate that LLL suppressed the activation of NF-κB signaling pathway in LPS treated MSCs through inhibiting phosphorylation of p65 and IκBα, which results in good anti-inflammatory effect. In addition, LLL attenuated activation-associated markers CD25 and CD69 in co-cultures of PBMC and 3 types of treated MSCs.

Low-Level Laser Effect on Proliferation, Migration, and Antiapoptosis of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been proved to be an important element in cell-based therapy. Photobiomodulation used extremely low-level lasers (LLLs) to affect the behavior of cells. The effect mechanism of LLLs on MSCs from human remained to be discovered. In this study, cell viability was assessed using MTS assays and cell cycle was evaluated by fluorescence-activated cell sorting (FACS). The influence of LLLs on mitochondrial biogenesis (fission or fusion) and function (ATP, reactive oxygen species [ROS], nitric oxide [NO]) was evaluated by transmission electron microscope, FACS, quantitative real time polymerase chain reaction (q-PCR), and immunocytochemistry. Cell migration and cytoskeleton alteration (actin and tubulin) were evaluated using transwell assay, immunocytochemistry, enzyme-linked immunosorbent assay, and western blotting. Cell apoptosis was evaluated using FACS, immunocytochemistry, and western blotting. We investigated that certain influence of LLLs on MSCs in vitro 6 or 24 h after 1 h of LLL irradiation. The mechanism of the effects included proliferation rate increase mediated by increased S phase proportion; mitochondrial biogenesis and function alteration mediated by fusion (Mfn1, Mfn2, and Opa-1) and fission (Fis1, Drp-1, and MTP18)-related proteins, NRF1, TFAM, PGC-1a, and upregulated intracellular ROS and NO concentration; migration acceleration through the ERK1/2 and FAK pathway and upregulation of growth factors such as HGF and PDGF; and resistance to apoptosis with increased Bcl-2 and decreased Bax, or through tunneling nanotube formation between LLL-treated MSCs and 5-fluorouracil-induced apoptotic MSCs. These observations suggested that LLLs enhanced stem cell survival and therapeutic function, which could appear to be an innovative pretreatment in the application of MSCs.

Lung tumor exosomes induce a pro-inflammatory phenotype in mesenchymal stem cells via NFκB-TLR signaling pathway.

BACKGROUND: In tumor microenvironment, a continuous cross-talk between cancer cells and other cellular components is required to sustain tumor progression. Accumulating evidence suggests that exosomes, a novel way of cell communication, play an important role in such cross-talk. Exosomes could facilitate the direct intercellular transfer of proteins, lipids, and miRNA/mRNA/DNAs between cells. Since mesenchymal stem cells (MSCs) can be attracted to tumor sites and become an important component of the tumor microenvironment, there is an urgent need to reveal the effect of tumor exosomes on MSCs and to further explore the underlying molecular mechanisms. METHODS: Exosomes were harvested from lung cancer cell line A549 and added to MSCs. Secretion of inflammation-associated cytokines in exosome-treated MSCs were analyzed by RT-PCR and ELISA. The growth-promoting effect of exosome-treated MSCs on lung tumor cells was evaluated by in vivo mouse xenograft model. Signaling pathway involved in exosomes-treated MSCs was detected by PCR array of human toll-like receptor signaling pathway, RT-PCR, and Western blot. RESULTS: Data showed that lung tumor cell A549-derived exosomes could induce a pro-inflammatory phenotype in MSCs named P-MSCs, which have significantly elevated secretion of IL-6, IL-8, and MCP-1. P-MSCs possess a greatly enhanced ability in promoting lung tumor growth in mouse xenograft model. Analysis of the signaling pathways in P-MSCs revealed a fast triggering of NF-κB. Genetic ablation of Toll-like receptor 2 (TLR2) by siRNA and TLR2-neutralizing antibody could block NF-κB activation by exosomes. We further found that Hsp70 present on the surface of lung tumor exosomes contributed to the induction of P-MSCs by A549 exosomes. CONCLUSIONS: Our studies suggest a novel mechanism by which lung tumor cell-derived exosomes induce pro-inflammatory activity of MSCs which in turn get tumor supportive characteristics.

Lung cancer exosomes initiate global long non-coding RNA changes in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can be attracted to tumor sites and become an important component of the tumor microenvironment, thus contributing to tumor development. Emerging evidence suggests that tumor cells could transfer genetic information into MSCs through the release of exosomes. However, the molecular mechanisms by which tumor exosomes contribute to interactions between MSCs and tumor cells remain largely unknown. In this study, we found that lung tumor cell derived exosomes could inhibit MSCs osteogenic and adipogenic differentiation. We then investigated the involvement of long non-coding RNAs, a new class of regulators, in tumor exosome treated MSCs by a comprehensive lncRNA and mRNA profiling. lncRNAs (9.1%) (2775 out of 30586) and 9.3% of protein-coding mRNA (2439 out of 26109) were differentially expressed (fold-change ≥2; P-value ≤0.05) in lung tumor cell exosome treated MSCs. Furthermore, we characterized the differentially expressed lncRNAs through their classes and length distribution and correlated them with differentially expressed mRNA. Noteworthy, GO analysis of biological process showed that upregulated mRNAs were enriched in mRNA metabolic process, while downregulated ones were enriched in detection of mechanical stimulus involved in sensory perception. Pathway analysis indicated that 32 pathways were upregulated while 7 were downregulated in A549 exosome treated MSCs. Here, we are the first to determine genome-wide lncRNA expression patterns in exosome treated MSCs by microarray and the results will bring new insights into the mechanisms underlying interactions between tumor cells exosomes and its environmental component the MSCs.

[Osteogenic and adipogenic differentiation of bone marrow multipotent mesenchymal stem cells in the mice with hemorrhagic anemia].

This study was aimed to investigate the effects of osteogenic and adipogenic differentiation of bone marrow multipotent mesenchymal stem cells (MSC) on hematopoiesis. A hemorrhagic anemia mouse model was established by exsanguinating of 0.3 ml blood from angular vein every 1-2 days for 4 weeks. The number of leukocytes, erythrocytes and neutrophils, hemoglobin level, ratio of reticulocyte in peripheral blood and bone marrow cell colony forming unit (CFU) were detected for the identification of the model. The differential potential of MSC in the hemorrhagic anemia mice were identified by CFU-F, histopathologic analysis, and osteogenesis and adipogenesis-related gene expression. The results showed that the erythrocyte numbers of peripheral blood and hemoglobin level decreased in the hemorrhagic anemia mice compared with the control, while the ratio of reticulocyte, the numbers of bone marrow cells and the CFU increased. Furthermore, the numbers of CFU-F, bone marrow hematopoietic cells, and osteogenic cells increased. However, the number of adipocytes decreased. Expressions of osteogenesis-related genes Runx2 and OSX were up-regulated, and adipogenesis-related genes aP2 and PPARγ2 were down-regulated in the hemorrhagic anemia mice compared with the control. It is concluded that the potential of osteogenic differentiation of MSC is enhanced, while the potential of adipogenic differentiation of MSC is weakened in the hemorrhagic anemia mice.

Hematopoietic recovery following chemotherapy is improved by BADGE-induced inhibition of adipogenesis.

This study was designed to investigate the role of increased adipocytes in the bone marrow (BM) niche induced by high-dose chemotherapy in hematopoietic recovery. Arabinosylcytosine (Ara-C) was administered to adult C57BL/6J mice to induce adipogenesis in the BM. We investigated the effects of adipogenesis on hematopoietic recovery following chemotherapy, using the peroxisome proliferator-activated receptor gamma inhibitor, bisphenol A diglycidyl ether (BADGE). Adipocyte hyperplasia could be induced by Ara-C treatment in BM and inhibited by BADGE. The accelerated recovery of leukocyte counts, increased colony forming units, and a higher proportion of Ki67(+)CD45(+) BM cells and Ki67(+)Lin(-)Sca1(+)c-kit(+) hematopoietic stem cells were observed in the long bone marrow of adipocyte-inhibited mice, as well as an increase in the number of CD45(+) BM cells in the tail fatty marrow compared to controls. Adipocytes participated in creating a distinctive niche for hematopoietic cells. In addition, lower expression of stromal cell-derived factor-1α and hypoxia-inducible factor-1 alpha were detected in the BADGE-treated group. These results indicate that hematopoietic recovery is improved following chemotherapy in adipogenesis-inhibited mice. In addition, adipocytes may create an individual niche that affects the proliferation and migration of hematopoietic cells in vitro and in vivo.

Stromal-derived factor-1 deficiency in the bone marrow of acute myeloid leukemia.

Chemokine stromal-derived factor-1 (SDF-1) and its receptor CXCR4 have been shown to play an important role in the migration and homing of the transplanted hematopoietic stem cells (HSCs). Mesenchymal stem cells (MSCs) express these molecules. This study is to test the hypothesis that acute myeloid leukemia (AML) alters the expression of SDF-1/CXCR4 in human bone marrow MSCs. Expression of both CXCR4 and SDF-1 was found to be increased, but excessively retained, in the MSCs in AML. In contrast, the SDF-1 level in bone marrow plasma and supernatant of cultured MSCs from AML patients were reduced, while the SDF-1 was able to efficiently induce a dose-dependent migration of MSCs in vitro. Our results demonstrate that altered expression and distribution of SDF-1/CXCR4 in MSCs may contribute to SDF-1 deficiency in the plasma of AML patients. The migration of MSCs may be negatively affected by the SDF-1 deficiency.