Synergistic effects of mesenchymal stem cell-derived extracellular vesicles and dexamethasone on macrophage polarization under inflammatory conditions.

The undesirable inflammation and the excessive M1 macrophage activity may lead to inflammatory diseases. Corticosteroids and stem cell therapy are used in clinical practice to promote anti-inflammatory responses. However, this protocol has limitations and is associated with numerous side effects. In this study, the synergistic anti-inflammatory effects of dexamethasone (Dex) and mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) were evaluated to enhance the polarization of M1 inflammatory macrophages into the anti-inflammatory (M2) phenotype. Hence, we designed different combinations of Dex and EVs using three methods, including EVs isolated from Dex-preconditioned MSCs (Pre-Dex-EVs), EVs loaded with Dex (L-Dex-EVs), and EVs and Dex co-administration (Dex + EVs). All designed EVs had a significant effect on reducing the expression of M1-related genes (iNOS, Stat1, and IRF5), cytokines (IL6 and TNF-a), and CD markers (CD86) in lipopolysaccharide-stimulated macrophages. On the other hand, these combinations promoted the expression of alternative-activated M2-related genes (Arg-1, Stat6, and IRF4), cytokine (IL10), and CD markers (CD206).The combination of Dex and MSC-EVs enhances the effectiveness of both and synergistically promotes the conversion of inflammatory macrophages into an anti-inflammatory state.

Immunophenotyping characteristics of COVID-19 patients: Peripheral blood CD8+ HLA-DR+ T cells as a biomarker for mortality outcome.

INTRODUCTION: The goal of this study was to identify biomarker(s) to assign risk of mortality in COVID-19 patients to improve intensive care unit (ICU) and coronary care unit  management. A total of 100 confirmed COVID-19 patients admitted at Imam Khomeini Hospital in Tehran, were compared to 70 control subjects. Peripheral blood leukocyte was studied using staining reagents included CD3, CD4, CD8, HLA-DR, CD19, CD16, and CD56. The immunophenotyping analysis was evaluated using the FACSCalibur instrument. To investigate the cell density of lung infiltrating T cells, postmortem slides of needle necropsies taken from the lung tissue of 3 critical patients were evaluated by immunohistochemistry staining. The number of lymphocyte subpopulations was significantly lower in COVID-19 patients than in the control group. Regarding the disease severity, the absolute count of T, NK, and HLA-DR+ T cells were significantly reduced in severe patients compared to the moderate ones. The critical patients had a significantly lower count of CD8-HLA-DR+ T cells than the moderate cases. Regarding the disease mortality, based on univariate analysis, the count of HLA-DR+ T, CD8- HLA-DR+ T, and CD8+ HLA-DR+ T cells was associated with mortality in COVID-19 patients. Receiver operating characteristic curve analysis showed the count of CD8+ HLA-DR+ T cells is the best candidate as a biomarker for mortality outcome. Furthermore, pulmonary infiltration of T cells in the lung tissue showed only slight infiltrations of CD3+ T cells, with an equal percentage of CD4+ and CD8+ T cell subpopulation in the lung tissue. These findings suggest that close monitoring of the value of CD8+ HLA-DR+ T cells in COVID-19 patients may be helpful to identify high-risk patients. However, further studies with larger sample size are needed.

In vitro treatment of murine splenocytes with extracellular vesicles derived from mesenchymal stem cells altered the mRNA levels of the master regulator genes of T helper cell subsets.

INTRODUCTION: The purpose of the current study was to evaluate the effect of mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) on the production of cytokines and expression of genes, which are corresponded to the subsets of T helper cells. MATERIALS AND METHODS: The supernatant of the second passage of MSCs that had been isolated from C57BL/6 mice abdominal adipose tissue was used to collect the MSC-EV. Splenocytes of healthy mice were activated using anti-CD3 and anti-CD28 antibodies and simultaneously were treated using the MSC-EVs. The proliferation rate of lymphocytes and the frequency of regulatory T cells were measured using flow cytometry. In addition, the expressions of T helper cell subset-specific transcription factors were evaluated using a real-time PCR assay. To appraise the effects of MSC-EV on splenocytes, the levels of IFN-γ, IL-17A, IL-10, and TGF-ß were measured using ELISA. RESULTS: The results showed that the treatment of the CD3/CD28-activated splenocytes with MSC-EV did not statistically change the proliferation of CD3+ splenocytes. However, after the treatment, the mRNA levels of Foxp3 and Elf4 as well as the frequency of regulatory T cells was significantly higher when compared to the control group. The expression levels of Gata3, Rorc, and Tbx21 were down-regulated while, the corresponding cytokines levels did not alter. CONCLUSION: The results revealed that the in vitro treatment of MSC-EV was associated with the increase in the frequency of CD4+CD25+FOXP3+ T cells and upregulation of Foxp3 mRNA level.

In vitro naive CD4+ T cell differentiation upon treatment with miR-29b-loaded exosomes from mesenchymal stem cells.

BACKGROUND: Gene regulation by microRNA (miRNA) is central in T lymphocytes differentiation processes. Here, we investigate miRNA-29b (miR-29b) roles in the reprogramming of T cell differentiation, which can be a promising therapeutic avenue for various types of inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. METHODS AND RESULTS: Adipose Mesenchymal Stem Cell-derived exosomes (AMSC-Exo) enriched with miR-29b were delivered into naive CD4+ T (nCD4+) cells. The expression level of important transcription factors including RAR-related orphan receptor gamma (RORγt), GATA3 binding protein (GATA3), T-box transcription factor 21, and Forkhead box P3 was determined by quantitative Real-Time PCR. Moreover, flow cytometry and Enzyme-linked Immunosorbent Assay were respectively used to measure the frequency of T regulatory cells and the levels of cytokines production (Interleukin 17, Interleukin 4, Interferon-gamma, and transforming growth factor beta. This study indicates that the transfection of miR-29b mimics into T lymphocytes through AMSC-Exo can alter the CD4+ T cells' differentiation into other types of T cells. CONCLUSIONS: In conclusion, AMSC-Exo-based delivery of miR-29b can be considered as a new fascinating avenue for T cell differentiation inhibition and the future treatment of several inflammatory disorders.

Regulation of the Th17/Treg balance by human umbilical cord mesenchymal stem cell-derived exosomes protects against acute experimental colitis.

Increasing evidence suggests that mesenchymal stem cells (MSCs) have immunosuppressive properties mediated by MSC-derived small extracellular vesicles (sEV). Exosomes are small extracellular vesicles that contain components that regulate immune cell function. We investigated the immunomodulatory effects of MSC-derived Exosome (MSC-Exo) on the severity of colitis using the dextran sulfate sodium (DSS)-induced colitis model. Exosomes were administrated intraperitoneally. Daily changes in body weight, stool consistency, and bleeding were assessed to determine the impact of MSC-Exos on colitis. Several measurements were taken, including the colon weight, length, and histological analysis of the colon tissues. The percentage of regulatory T cells and IL-10, TGF-ß, IL-17, TNF-α, and IFN-γ levels were calculated in the mesenteric lymph node (MLN) and spleen. The results showed MSC-Exos improved clinical manifestations of colitis. Colon macroscopic and histological observations also showed improvement in tissue destruction. The results illustrated that MSC-Exos might attenuate colitis by regulating Treg/Th17 balance, increasing anti-inflammatory, and decreasing pro-inflammatory cytokines expression. As a result, MSC-Exos could be used as an immunomodulatory approach to treating bowel inflammation.

Exosomes derived from rapamycin-treated 4T1 breast cancer cells induced polarization of macrophages to M1 phenotype.

M2 macrophages are the most prevalent type in the tumor microenvironment and their polarization to M1 type can be used as a potential cancer immunotherapy. Here, we investigated the role of tumor microenvironment and particularly purified exosomes in M2 to M1 macrophage polarization. Rapamycin treatment on triple-negative breast cancer cells (TNBC) was performed. Tumor cells-derived exosomes (called texosomes) were isolated and characterized using scanning electron microscopy, transmission electron microscopy, dynamic light scattering, high-performance liquid chromatography, Fourier transform infrared, and Western blot assays. M2 mouse peritoneal macrophages were treated with rapamycin or rapamycin-texosome. Then, M1/M2 phenotype-specific marker genes and proteins were measured to assess the degree of M2 to M1 polarization. Finally, nitric oxide (NO) production, phagocytosis, and efferocytosis assays were assessed to verify the functionality of the polarized macrophages. Purified rapamycin-texosomes significantly increased the expression of the M1 markers (Irf5, Nos2, and CD86) and decreased M2 markers (Arg, Ym1, and CD206). In addition, the levels of M1-specific cytokines tumor necrosis factor alpha and interleukin 1ß (IL-1ß) were increased, whereas the levels of M2 specific cytokines IL-10 and transforming growth factor beta were declined. Furthermore, texosome treatment increased NO concentration and phagocytosis and decreased efferocytosis indicating M1 polarization. These findings suggest rapamycin-texosomes can induce M2 to M1 macrophages polarization as a potential immunotherapy for TNBC.

Coculture of adipose-derived mesenchymal stem cells/macrophages on decellularized placental sponge promotes differentiation into the osteogenic lineage.

BACKGROUND: Several factors like three-dimensional microstructure, growth factors, cytokines, cell-cell communication, and coculture with functional cells can affect the stem cells behavior and differentiation. The purpose of this study was to investigate the potential of decellularized placental sponge as adipose-derived mesenchymal stem cells (AD-MSCs) and macrophage coculture systems, and guiding the osteogenic differentiation of stem cells. METHODS: The decellularized placental sponge (DPS) was fabricated, and its mechanical characteristics were evaluated using degradation assay, swelling rate, and pore size determination. Its structure was also investigated using hematoxylin and eosin staining and scanning electron microscopy. Mouse peritoneal macrophages and AD-MSCs were isolated and characterized. The differentiation potential of AD-MSCs co-cultured with macrophages was evaluated by RT-qPCR of osteogenic genes on the surface of DPS. The in vivo biocompatibility of DPS was determined by subcutaneous implantation of scaffold and histological evaluations of the implanted site. RESULTS: The DPS had 67% porosity with an average pore size of 238 µm. The in vitro degradation assay showed around 25% weight loss during 30 days in PBS. The swelling rate was around 50% during 72 h. The coculture of AD-MSCs/macrophages on the DPS showed a significant upregulation of four differentiation osteogenic lineage genes in AD-MSCs on days 14 and 21 and a significantly higher mineralization rate than the groups without DPS. Subcutaneous implantation of DPS showed in vivo biocompatibility of scaffold during 28 days follow-up. CONCLUSIONS: Our findings suggest the decellularized placental sponge as an excellent bone substitute providing a naturally derived matrix substrate with biostructure close to the natural bone that guided differentiation of stem cells toward bone cells and a promising coculture substrate for crosstalk of macrophage and mesenchymal stem cells in vitro.

Exosome engineering in cell therapy and drug delivery.

Cell-derived exosomes have opened new horizons in modern therapy for advanced drug delivery and therapeutic applications, due to their key features such as low immunogenicity, high physicochemical stability, capacity to penetrate into tissues, and the innate capacity to communicate with other cells over long distances. Exosome-based liquid biopsy has been potentially used for the diagnosis and prognosis of a range of disorders. Exosomes deliver therapeutic agents, including immunological modulators, therapeutic drugs, and antisense oligonucleotides to certain targets, and can be used as vaccines, though their clinical application is still far from reality. Producing exosomes on a large-scale is restricted to their low circulation lifetime, weak targeting capacity, and inappropriate controls, which need to be refined before being implemented in practice. Several bioengineering methods have been used for refining therapeutic applications of exosomes and promoting their effectiveness, on the one hand, and addressing the existing challenges, on the other. In the short run, new diagnostic platforms and emerging therapeutic strategies will further develop exosome engineering and therapeutic potential. This requires a thorough analysis of exosome engineering approaches along with their merits and drawbacks, as outlined in this paper. The present study is a comprehensive review of novel techniques for exosome development in terms of circulation time in the body, targeting capacity, and higher drug loading/delivery efficacies.

Evaluating the effect of LPS from periodontal pathogenic bacteria on the expression of senescence-related genes in human dental pulp stem cells.

The human dental pulp stem cells (hDPSCs) are one of the readily available sources of multipotent mesenchymal stem cells (MSCs) and can be considered as a type of tool cells for cell-based therapies. However, the main limitation in the clinical use of these cells is DPSC senescence, which can be induced by lipopolysaccharide (LPS) of oral pathogenic bacteria. Up to now, far little attention has been paid to exploring the molecular mechanisms of senescence in DPSCs. So, the current study aimed to investigate the underlying molecular mechanism of senescence in hDPSCs stimulated with Porphyromonas gingivalis (P. gingivalis) and Escherichia coli (E. coli)-derived LPSs, by evaluating both mRNA and protein expression of four important senescence-related genes, including TP53, CDKN1A, CDKN2A and SIRT1. To this purpose, hDPSCs were stimulated with different LPSs for 6, 24 and 48 h and then the gene expression was evaluated using quantitative real-time polymerase chain reaction (qPCR) and western blotting. Following stimulation with P. gingivalis and E. coli-derived LPSs, the relative mRNA and protein expression of all genes were significantly up-regulated in a time-dependent manner, as compared with unstimulated hDPSCs. Moreover, the hDPSCs stimulated with P. gingivalis LPS for 6 and 24 h had the highest mRNA expression of CDKN1A and SIRT1, respectively (p < 0.0001), whereas the highest mRNA expression of CDKN2A and TP53 was seen in hDPSCs stimulated with E. coli LPS for 48 h (p < 0.0001). In summary, because DPSCs have been reported to have therapeutic potential for several cell-based therapies, targeting molecular mechanisms aiming at preventing DPSC senescence could be considered a valuable strategy.

Adipose-derived mesenchymal stem cell-secreted exosome alleviates dextran sulfate sodium-induced acute colitis by Treg cell induction and inflammatory cytokine reduction.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is an inflammatory condition that results in gastrointestinal tract damage. Various factors, including environmental and genetic agents, disrupt the function of the intestinal immune system that can lead to IBD. Mesenchymal stem cells (MSCs) display an immunoregulatory function and demonstrate regenerative potential by paracrine action. In this study, we evaluated the immunomodulatory effects of MSCs' derived exosomes in the acute form of dextran sulfate sodium (DSS)-induced colitis. Exosomes were isolated from adipose-derived MSCs. Acute colitis was induced by DSS. The exosome was used by intraperitoneal injection into mice with acute colitis. Stool consistency, body weight changes, bleeding severity, colon length, and weight were examined. At the experimental endpoint (Day 7), the changes in the colon tissue were evaluated. The level of cytokines of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), IL-4, IL-12, transforming growth factor-ß (TGF-ß) and, IL-10, and Treg cells percentage were assayed. Results showed that exosome administration diminished colon shortening, bodyweight loss, bleeding, and colon injury. The levels of IFN-γ, TNF-α, IL-12, and IL-17 were decreased, and the level of TGF-ß, IL-4, and IL-10 were increased in lymph node and spleen of mice treated with exosome.  Percentages of CD4+ CD25+ Foxp3+ Treg cells were grown in the lymph node and spleen of mice treated with exosomes. Overall, current data suggest that MSC-derived exosome could regulate the Treg population and improves inflammation in DSS-induced acute colitis.

In vitro and in vivo evaluation of anti-tumoral effect of M1 phenotype induction in macrophages by miR-130 and miR-33 containing exosomes.

In the tumor microenvironment, macrophages polarize into the M2 phenotype to facilitate tumorigenesis. Tumor-derived exosomes can act as mediators between the tumor microenvironment and stromal cells by transporting proteins, mRNAs, and miRNAs. Exosomal miRNAs play a pivotal role in modulating tumor microenvironment and macrophage polarization. Here, we overexpressed miR-130 and miR-33 in exosomes of MDA-MB-231 cells and investigated their effect on macrophage polarization and tumor progression. For this purpose, exosomes were extracted from MDA-MB-231 cells and characterized using dynamic light scattering, electron microscopy, and western blotting of exosomal markers. Then, miR-130 or miR-33 containing exosomes were used to treat IL4-induced M2 or tumor-associated macrophages (TAMs). After treatment, the polarization status of macrophages, including the expression of M1 specific genes, and the secretion of cytokines were evaluated. Finally, the conditioned medium from exosome-treated macrophages was incubated with cancer cells to evaluate its effect on the migration and invasion ability of cancer cells and, in vivo experiments investigated the effect of exosome-treated macrophages on breast cancer progression. Exosomes characterization results approved the range of size and homogeneity of extracted exosomes. Overexpression of miR-130 and miR-33 in exosomes increased the expression of M1 signature genes (IRF5, MCP1, CD80) and secretion of cytokines (IL-1ß and TNF-α) as well as yeast phagocytic activity of macrophages. Besides, the conditioned medium of macrophages treated with miRNA containing exosomes declined the migration and invasion ability of cancer cells. The in vivo results indicated the inhibitory effect of exosome-treated macrophages on tumor growth. Furthermore, the results showed that in response to exosome-treated macrophages, the production of TNF-α by spleen cells increased, while the production of IL-10 and TGF-ß by these cells decreased. These findings suggest that overexpression of miR-130 and miR-33 in exosomes can decrease tumor progression by shifting macrophage polarization from M2 to M1 phenotype and can be a potential therapeutic strategy for tumor interventions.

Hyperglycemia results in decreased immune cell infiltration and increased viral load in the lung in a mouse model of RSV infection.

Respiratory Syncytial virus (RSV) infection is a feared disease in vulnerable populations with impaired immune responses. There is currently no vaccine against RSV and young children along with elderly people are at increased risk of severe or sometimes life-threatening RSV infection. Hyperglycemia with immunomodulatory patterns can impact on infectious disease outcomes and immune system responses in diabetic patients. Even though research continues to uncover the complex mechanisms underlying RSV immunopathogenesis and diabetes mellitus disease separately, limited information is available about interaction between these two phenomena. Here, we evaluated the influence of hyperglycemia as the hallmark of diabetes mellitus disease on the pathogenesis and immunopathogenesis of RSV in a mouse model. In this experiment, hyperglycemia was induced by intraperitoneal injection of Streptozotocin (STZ), and after diabetes confirmation, mice were infected with RSV-A2, and the immune responses were followed for 5 days until the mice were sacrificed. Analyses on airway immune cell influx, T-Lymphocyte subtypes, cytokines secretion, lung histopathology, and viral load were conducted. Our results showed that hyperglycemia resulted in reduced lung immune cells infiltration totally and it was associated with decreased pathological damage of the lung. Following RSV infection in hyperglycemic mice, the ratio of CD4/CD8 T-Lymphocytes due to CD8+ depletion, increased. Furthermore, the level of IFN-γ and IL-17A cytokines decreased, whereas IL-10 showed an upward trend and the viral load increased in hyperglycemic mice compared with normoglycemic mice. In conclusion, these findings indicate that hyperglycemia can ameliorate and downregulate RSV-induced inflammatory and antiviral responses, and result in increment of viral load.

Wharton's Jelly Mesenchymal Stem Cells Exosomes and Conditioned Media Increased Neutrophil Lifespan and Phagocytosis Capacity.

Neutrophils are the first cells involved in inflammation and pathogen elimination, but they have a short lifespan. So, strategies for enhancing neutrophil lifespan and activities can be useful in many situations such as patients with immunodeficiencies. Previous researches demonstrated that mesenchymal stem cell (MSC) has anti-apoptotic effects on neutrophils. These multipotent cells have immunomodulatory properties and can be isolated from different tissues. MSCs isolated from Wharton's jelly (WJ-MSCs), a mucosal connective tissue of the umbilical cord, may be better candidates than MSCs obtained from bone marrow or adipose tissue, because WJ-MSCs are younger and protected from damages that are resulted from aging, environmental toxins, and diseases. In addition, they have high proliferative capacity, easier accessibility, and more abundance. It was shown that following in vitro expansion, they are more effective than other sources of MSCs. Cell to cell contact or secretion of soluble factors and exosomes are the main approaches of MSCs in applying their effects. Exosomes and conditioned media (CM) were prepared from WJ-MSCs. Then, neutrophils were isolated and cultured with medium, CM, or exosomes. Then, neutrophil respiratory burst, apoptosis, and phagocytosis capacity were assessed by NBT assay, Annexin V-PI method, and Giemsa staining, respectively. Both treatments improved neutrophil lifespan and phagocytosis. Only MSC-CM could enhance neutrophil respiratory burst. This research demonstrated that MSC-exosomes and CM have protective effects on neutrophil function and lifespan. It can be concluded that MSC mediators can be responsible factors for protective functions of MSCs on neutrophils.

Nationwide population-based surveys of Iranian COVID-19 Serological Surveillance (ICS) program: The surveys protocol.

Background: Serological surveillance of COVID-19 through conducting repetitive population-based surveys can be useful in estimating and monitoring changes in the prevalence of infection across the country. This paper presents the protocol of nationwide population-based surveys of the Iranian COVID-19 Serological Surveillance (ICS) program. Methods: The target population of the surveys is all individuals ≥6 years in Iran. Stratified random sampling will be used to select participants from those registered in the primary health care electronic record systems in Iran. The strata are the 31 provinces of the country, in which sampling will be done through simple random sampling. The sample size is estimated 858 individuals for each province (except for Tehran province, which is 2574) at the first survey. It will be recalculated for the next surveys based on the findings of the first survey. The participants will be invited by the community health workers to the safe blood sampling centers at the district level. After obtaining written informed consent, 10 mL of venous blood will be taken from the participants. The blood samples will be transferred to selected reference laboratories in order to test IgG and IgM antibodies against COVID-19 using an Iranian SARS-CoV-2 ELISA Kit (Pishtaz Teb). A serologically positive test is defined as a positive IgG, IgM, or both. After adjusting for the measurement error of the laboratory test, nonresponse bias, and sampling design, the prevalence of COVID-19 will be estimated at the provincial and national levels. Also, the approximate incidence rate of infection will be calculated based on the data of both consecutive surveys. Conclusion: The implementation of these surveys will provide a comprehensive and clear picture of the magnitude of COVID-19 infection and its trend over time for health policymakers at the national and subnational levels.

Comparative immunomodulatory properties of mesenchymal stem cells derived from human breast tumor and normal breast adipose tissue.

OBJECTIVE: Mesenchymal stem cells (MSCs), one of the most important stromal cells in the tumor microenvironment, play a major role in the immunomodulation and development of tumors. In contrast to immunomodulatory effects of bone marrow-derived MSCs, resident MSCs were not well studied in tumor. The aim of this study was to compare the immunomodulatory properties and protein secretion profiles of MSCs isolated from breast tumor (T-MSC) and normal breast adipose tissue (N-MSC). MATERIALS AND METHODS: T-MSCs and N-MSCs were isolated by the explant culture method and characterized, and their immunomodulatory function was assessed on peripheral blood lymphocytes (PBLs) by evaluating the effects of MSC conditioned media on the proliferation and induction of some cytokines and regulatory T cells (Tregs) by BrdU assay, ELISA, and flow cytometry. In addition, we compared the secretion of indoleamine 2,3-dioxygenase (IDO), vascular endothelial growth factor (VEGF), matrix metallopeptidase (MMP)-2, MMP-9, and Galectin-1. RESULTS: T-MSCs showed a higher secretion of transforming growth factor beta (TGF-ß), prostaglandin E2 (PGE2), IDO, and VEGF and lower secretion of MMP-2 and MMP-9 compared with N-MSCs. However, no significant difference was found in the secretion of interferon gamma (IFN-γ), interleukin 10 (IL10), IL4, IL17, and Galectin-1 in T-MSCs and N-MSCs. The immunomodulatory effect of soluble factors on PBLs showed that T-MSCs, in contrast to N-MSCs, stimulate PBL proliferation. Importantly, the ability of T-MSCs to induce IL10, TGF-ß, IFN-γ, and PGE2 was higher than that of N-MSCs. In addition, T-MSCs and N-MSCs exhibited no significant difference in Treg induction. CONCLUSION: MSCs educated in stage II breast cancer and normal breast adipose tissue, although sharing a similar morphology and immunophenotype, exhibited a clearly different profile in some immunomodulatory functions and protein secretions.

MicroRNA-218 competes with differentiation media in the induction of osteogenic differentiation of mesenchymal stem cell by regulating ß-catenin inhibitors.

Osteoporosis, a systemic skeletal disorder specified by low bone mass, is associated with bone fragility and the raised risk of fractures. Activation of the Wnt/ß-catenin signaling pathway has been directly demonstrated as a prominent biological event in the prevention of osteoporosis. Recently, critical roles of microRNAs (miRNAs) were further revealed in Wnt/ß-catenin signaling activation and thereby contributing to the development and maintenance of the human skeleton. In this study, we investigated whether miR-218 can significantly promote the osteogenic differentiation of mesenchymal stem cells in conditional media by regulating ß-catenin signaling inhibitors. The pre-miRNA nucleotide sequence of miR-218 was cloned into the pEGP-miR vector. Next, human adipose tissue-derived mesenchymal stem cells (AD-MSCs) were isolated, characterized, and transfected using pEGP-miR-218.Subsequently, the osteogenic potential of AD-MSCs was investigated in different treated groups using alkaline phosphatase (ALP)activity, calcium mineral deposition, and the expression of osteogenesis-related genes. Finally, negative regulators of Wnt signaling targeted by miR-218 were bioinformatically predicted. Our results indicated a significant increase in the ALP activity, mineralization, and osteogenesis-related genes expression in the AD-MSCs transfected with pEGP-miR-218. Also, the bioinformatic surveys and gene expression results showed that adenomatosis polyposis coli (APC) and glycogen synthase kinase 3 (GSK3-ß) were downregulated in the transfected AD-MSCs in both differential and conditional media. This study provided evidence that miR-218 can promote osteogenic differentiation of AD-MSCs even in conditional media. Therefore, our findings suggest miR-218 as a putative novel therapeutic candidate in the context of osteoporosis and other bone metabolism-related diseases.

A novel natural antisense transcript at human SOX9 locus is down-regulated in cancer and stem cells.

OBJECTIVE: SOX9 is a key transcription factor with important roles in regulating proliferation and differentiation of various cell types. Dysregulation of SOX9 expression has been involved with pathogenesis of different developmental, degenerative, and neoplastic disorders. Natural antisense transcripts (NATs) are long non-coding RNAs with increasing significance in regulation of gene expression. However, the presence of a NAT at SOX9 locus has been so far unclear. RESULT: We detected a natural antisense transcript at SOX9 locus (SOX9-NAT) through strand-specific RT-PCR. In contrast to SOX9 sense RNA (mRNA), SOX9-NAT was down-regulated in cancer tissues and cell lines compared with their normal counterparts. In addition, reciprocal to SOX9 mRNA, SOX9-NAT was also down-regulated in human embryonic stem cells in comparison with human fibroblasts in vitro. CONCLUSION: The negative correlation between SOX9 mRNA and SOX9-NAT was confirmed by analyzing qPCR data, as well as RNA-Seq datasets of several human cancers. Our data suggest a functional role for SOX9-NAT in the regulation of SOX9 mRNA as a potential target in cancer treatment and regenerative medicine.

Investigating the route of administration and efficacy of adipose tissue-derived mesenchymal stem cells and conditioned medium in type 1 diabetic mice.

Type 1 diabetes (T1D) is a chronic autoimmune disease destroying the insulin-producing beta cells. Recently, stem cell therapy has been tested to treat T1D. In the present study, we aim to investigate the effects of intraperitoneal and intravenous infusion of multipotent mesenchymal stem/stromal cells (MSCs) and MSC-conditioned medium (MSC-CM) in an experimental model of diabetes, induced by multiple injections of Streptozotocin (STZ). The adipose tissue-derived MSC and MSC-CM were isolated from C57Bl/6 male mice and characterized. Later, MSC and MSC-CM were injected intraperitoneally or intravenously into mice. The blood glucose, urinary glucose, and body weight were measured, and the percentages of CD4+ CD25+ FOXP3+ T cells as well as the levels of IFN-γ, TGF-ß, IL-4, IL-17, and IL-10 were evaluated. Our results showed that both intraperitoneal and intravenous infusions of MSC and MSC-CM could decrease the blood glucose, recover pancreatic islets, and increase the levels of insulin-producing cells. Furthermore, the percentage of CD4+ CD25+ FOXP3+ T cells was increased after intraperitoneal injection of MSC or MSC-CM and intravenous injection of MSCs. After intraperitoneal injection of the MSC and MSC-CM, the levels of inflammatory cytokines reduced, while the levels of anti-inflammatory cytokines increased. Together current data showed that although both intraperitoneal and intravenous administration had beneficial effects on T1D animal model, but intraperitoneal injection of AD-MSC and AD-MSC-CM was more effective than systemic administration.

Generation of insulin-producing hepatocyte-like cells from human Wharton's jelly mesenchymal stem cells as an alternative source of islet cells.

Islet cell transplantation, as a treatment of type 1 diabetes, has a lot of complexity such as allograft rejections and an insufficient number of donors. The liver can be used as a replacement for endogenous insulin production. Hepatocytes can inherently respond to glucose levels and secrete proteins. Utilization of mesenchymal stem cells for curing diabetes represents a major focus of recent investigations. As a new choice for transplantation, we have proposed glucose-regulated insulin-producing hepatocyte-like cells, which produce insulin dependent on glucose levels. We have transfected human Wharton's jelly mesenchymal stem cells with the special construct, which included homology arms and glucose-responsive elements upstream of the minimum liver-type pyruvate kinase promoter-directed insulin gene. Then, we have differentiated these transfected cells to hepatocyte-like cells by using serial exposure of different inducing material and exogenous growth factors. Immunofluorescence analyses have demonstrated the expression of albumin, cytokeratin-18, Hep-Par1, α-fetoprotein, and insulin. The expression of hepatocyte marker genes in the differentiated cells was confirmed by reverse-transcription polymerase chain reaction. Interestingly, flow cytometry results showed that approximately 60% of the insulin-producing hepatocyte-like cells were simultaneously cytochrome P450 3A4 (CYP3A4) and insulin positive. CYP3A4 is a significant enzyme found in mature liver tissue. This confirmed that the differentiation and the transfection procedures were done correctly. They were functionally active by releasing insulin in response to elevated glucose concentrations in vitro. These applicable cells could be used in the liver for cell therapy of diabetes.

Effect of mesenchymal stem cells-derived exosomes on tumor microenvironment: Tumor progression versus tumor suppression.

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into different cell types. Owing to their immunosuppressive and anti-inflammatory properties, they are widely used in regenerative medicine, but they have a dual effect on cancer progression and exert both growth-stimulatory or -inhibitory effects on different cancer types. It has been proposed that these controversial effects of MSC in tumor microenvironment (TME) are mediated by their polarization to proinflammatory or anti-inflammatory phenotype. In addition, they can polarize the immune system cells that in turn influence tumor progression. One of the mechanisms involved in the TME communications is extracellular vesicles (EVs). MSCs, as one of cell populations in TME, produce a large amount of EVs that can influence tumor development. Similar to MSC, MSC-EVs can exert both anti- or protumorigenic effects. In the current study, we will investigate the current knowledge related to MSC role in cancer progression with a focus on the MSC-EV content in limiting tumor growth, angiogenesis, and metastasis. We suppose MSC-EVs can be used as safe vehicles for delivering antitumor agents to TME.