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.

Adipose derived mesenchymal stem cell exosomes loaded with miR-10a promote the differentiation of Th17 and Treg from naive CD4+ T cell.

AIMS: The balance between various CD4+ T cell subsets through highly regulated differentiation of naïve T cells is critical to ensure proper immune response, disruption of which may cause autoimmunity and cancers. miR-10a has been reported to regulate the fate of naïve T cells. Mesenchymal stem cells (MSC) derived exosomes are known effective immunomodulators and ideal vehicles for delivery of microRNAs. This study was aimed to examine the impacts of miR-10a on CD4+ cell fate upon exosomal delivery in combination with immunomodulatory effects of MSCs. MAIN METHODS: Exosomes isolated form adipose tissue derived mesenchymal stem cells (AD-MSC-Exo) were transfected with miR-10a and added to naïve T cells purified from mouse spleen. AD-MSC-Exos were characterized and the efficacy of miR-10a delivery was evaluated. The expression levels of T-bet, GATA3, RORγt, and Foxp3 and the secreted levels of IFN-γ, IL-4, IL-17, and TGF-ß respectively specific to Th1, Th2, Th17 and Treg, were assessed by qPCR and ELISA. KEY FINDINGS: Being transferred by AD-MSC-Exo, miR-10a was effectively induced in CD4+ T cells. Upon treatment with miR-10a loaded exosomes, the expression levels of RORγt and Foxp3 were enhanced and that of T-bet was reduced. Similarly, the secreted levels of IL-17, and TGF-ß were increased and that of IFN-γ was decreased. SIGNIFICANCE: Our data indicate that miR-10a loaded exosomes, promote Th17 and Tregs response while reduce that of Th1. Promotion of both Th17 and Tregs in concert, mediated by the combined effect of miR-10a and MSC-Exo, indicate new therapeutic potentials, particularly in line with novel anti-tumor immunotherapeutic strategies.

Inhibition of miR-34a reduces cellular senescence in human adipose tissue-derived mesenchymal stem cells through the activation of SIRT1.

AIMS: Human adipose derived mesenchymal stem cells (hAD-MSCs) as the most promising target for cell therapy and regenerative medicine, face senescence as a major drawback resulting in their limited proliferation and differentiation potentials. To evaluate the efficacy of miR-34a silencing as an anti-senescence strategy in hAD-MSCs, in this study common hallmarks of senescence were assessed after transient inhibition of miR-34a in hAD-MSCs. MATERIALS AND METHODS: The expression levels of miR-34a in hAD-MSCs at different passages were evaluated by real-time quantitative PCR. hAD-MSCs at passage 2 and passage 7 were transfected with miR-34a inhibitor. Doubling time assay, colony forming assay, and cell cycle analysis were performed to evaluate cell proliferation rate. The activity of senescence associated ß-galactosidase (SA-ß-gal) was assessed by histochemical staining. Moreover, the senescence associated molecular alterations including that of pro-senescence (P53, P21 and P16) and anti-senescence (SIRT1, HTERT and CD44) genes were examined by quantitative RT-PCR and western blot assays. To evaluate the differentiation potentials of MSCs, following adipogenic and osteogenic induction, the expression levels of lineage specific markers were analyzed by qPCR. KEY FINDINGS: Our results showed that inhibition of miR-34a enhances the proliferation, promotes the adipogenic and osteogenic differentiation potency, reduces the senescence associated-ß gal activity, and reverses the senescence associated molecular alterations in hAD-MSCs. SIGNIFICANCE: In this study, we showed that inhibition of miR-34a reduces the cellular senescence through the activation of SIRT1. Our findings support the silencing of miR-34a as an anti-senescence approach to improve the therapeutic potentials of hAD-MSCs.

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.

Rn7SK small nuclear RNA is involved in cellular senescence.

Rn7SK is a conserved small nuclear noncoding RNA which its function in aging has not been studied. Recently, we have demonstrated that Rn7SK overexpression reduces cell viability and is significantly downregulated in stem cells, human tumor tissues, and cell lines. In this study, we analyzed the role of Rn7SK on senescence in adipose tissue-derived mesenchymal stem cells (AD-MSCs). For this purpose, Rn7SK expression was downregulated and upregulated via transfection and transduction, respectively, in AD-MSCs and subsequently, various distinct characteristics of senescence including cell viability, proliferation, colony formation, senescence-associated ß galactosidase activity, and differentiation potency was analyzed. Our results demonstrated the transient knockdown of Rn7SK in MSCs leads to delayed senescence, while its overexpressions shows opposite effects. When osteogenic differentiation was started, however, they exhibited a greater differentiation potential than the original MSCs, suggesting a potential tool for stem cell-based regenerative medicine.

A simple and highly efficient method for transduction of human adipose-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into a wide range of cell types and provide a potential to transfer therapeutic protein in vivo, making them valuable candidates for gene therapy and cell therapy. However, using MSCs in in vivo is limited due to the low rate of transfection and transduction efficacy. Therefore, developing methods to efficiently transfer genes into MSCs would provide a number of opportunities for using them in the clinic. Here, we introduce a simple and robust method for efficient transduction of human adipose-derived MSCs by modification under the culture condition of human embryonic kidney cells 293 (HEK293T) and MSCs. Moreover, as a transduction enhancer, polybrene was replaced with Lipofectamine, a cationic lipid. Therefore, we showed that transduction of primary cells can be increased efficiently by modifying the culture condition.

Effect of efferocytosis of apoptotic mesenchymal stem cells (MSCs) on C57BL/6 peritoneal macrophages function.

Phagocytic clearance of apoptotic cells (Efferocytosis) could affect the polarization of macrophages and promote M2 anti-inflammatory and regulatory phenotype and function. Here we tested the hypothesis that efferocytosis of apoptotic Adipose-Derived Mesenchymal Stem Cells (AD-MSCs) promotes macrophage M2 polarization. In this study, Macrophages were incubated with apoptotic MSCs and after 48 h interleukin-10 (IL-10), transforming growth factor-alpha (TNFα), and nitric oxide (NO) production were measured. Furthermore, phagocytosis ability and arginase activity were analyzed. The results showed that apoptotic MSCs could reduce TNFα and NO production, and increase IL-10 levels. Moreover, arginase activity and phagocytosis ability were also increased in tested macrophages compared to controls. In Conclusion efferocytosis of AD-MSCs can alter the macrophages phenotype toward regulatory and anti-inflammatory phenotype.

Cell type-dependent functions of microRNA-92a.

The role of miR-17/92 family in development and progression of various cancers has been established. The members of this miRNA family have been shown to be over expressed and target various genes within proliferation, metastasis and angiogenesis pathways. Although all members might be overexpressed in a certain cancer type, only certain members of the family may have roles in progression of that cancer. In this study, we have chosen miR-92a, a member of the miR-17/92 family to compare its function in three different cancer cell lines. HL60, MCF7, and Jurkat cell lines were transduced with miR-92a and proliferation and apoptosis was measured in these cells by cell count, MTT, and caspase assays. Although in comparison to pre-miR-17/92, the level of miR-92a is higher in Jurkat cells compared to MCF7 and HL60 cells, here we have shown that increasing miR-92a levels results in apoptosis in Jurkat cells and proliferation in MCF7 and HL60 cells. miR-92a was also microinjected into mice fertilized eggs and after dissection, apoptosis was only observed in white pulp of spleen that is mainly made up of white blood cells. Our results show that miR-92a possesses a cell-type dependent function.