Cytochalasin B-Induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing IL2 Are Able to Stimulate CD8+ T-Killers to Kill Human Triple Negative Breast Cancer Cells

Interleukin 2 (IL2) was one of the first cytokines used for cancer treatment due to its ability to stimulate anti-cancer immunity. However, recombinant IL2-based therapy is associated with high systemic toxicity and activation of regulatory T-cells, which are associated with the pro-tumor immune response. One of the current trends for the delivery of anticancer agents is the use of extracellular vesicles (EVs), which can carry and transfer biologically active cargos into cells. The use of EVs can increase the efficacy of IL2-based anti-tumor therapy whilst reducing systemic toxicity. In this study, human adipose tissue-derived mesenchymal stem cells (hADSCs) were transduced with lentivirus encoding IL2 (hADSCs-IL2). Membrane vesicles were isolated from hADSCs-IL2 using cytochalasin B (CIMVs-IL2). The effect of hADSCs-IL2 and CIMVs-IL2 on the activation and proliferation of human peripheral blood mononuclear cells (PBMCs) as well as the cytotoxicity of activated PBMCs against human triple negative cancer MDA-MB-231 and MDA-MB-436 cells were evaluated. The effect of CIMVs-IL2 on murine PBMCs was also evaluated in vivo. CIMVs-IL2 failed to suppress the proliferation of human PBMCs as opposed to hADSCs-IL2. However, CIMVs-IL2 were able to activate human CD8+ T-killers, which in turn, killed MDA-MB-231 cells more effectively than hADSCs-IL2-activated CD8+ T-killers. This immunomodulating effect of CIMVs-IL2 appears specific to human CD8+ T-killer cells, as the same effect was not observed on murine CD8+ T-cells. In conclusion, the use of CIMVs-IL2 has the potential to provide a more effective anti-cancer therapy. This compelling evidence supports further studies to evaluate CIMVs-IL2 effectiveness, using cancer mouse models with a reconstituted human immune system.

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Biomimetic Membrane Vesicles: Evaluation of Inflammatory Response In Vivo

Blood ◽

10.1182/blood-2020-135826 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-1

Author(s):

Olga Vasileva ◽

Ekaterina E Garanina ◽

Albert A Rizvanov ◽

Marina Gomzikova

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Russian Federation ◽

Cytochalasin B ◽

Membrane Vesicles ◽

Host Cells ◽

Vector System ◽

Gm Csf ◽

Biomimetic Membrane ◽

The Russian Federation

Introduction: Biomimetic membrane vesicles are produced from live cells using cytochalasin B which disrupts the structure of the cytoskeleton and facilitate generation of membrane vesicles under subsequent vortexing. Membrane vesicles are pinched off from the cell surface, surrounded by a cytoplasmic membrane and contain the cytoplasm of parental cell. It is known that mesenchymal stem cells (MSCs) are immunoprivileged. The aim of our study was to determine whether Cytochalasin B-induced membrane vesicles (CIMVs) derived from mesenchymal stem cells retain the immunoprivileged properties of parental cells. Method: All experiments were carried out in compliance with the procedure protocols approved by Kazan Federal University and local ethics committee (protocol #5, date 27.05.2014). To analyze the immunogenicity, murine MSCs (7.5x104 cells) either CIMVs-MSCs (15µg) were injected i.v. in 8 week old mice (Mus musculus, C57Bl/6). CIMVs were used at a concentration equivalent to 7.5×104 MSCs based on total protein concentration. Serum isolation was performed after 2 hours post-administration. Secretion of inflammatory cytokines was evaluated using multiplex analysis BioPlex Pro Mouse 23 Plex kit. (BioRad, USA). Results: We detected all investigated cytokines in serum of control and experimental mice: Eotaxin, G-CSF, GM-CSF, IFN-g, IL-10, IL-13, IL-17A, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL12p40, KC, MCP-1, MIP-1a, MIP-1b, RANTES, TNFa. Allogenic MSCs but not CIMVs increased concentration of Eotaxin, G-CSF, IL-17A and IL-9. The level of GM-CSF, IFN-g, IL-10, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL12p40, KC, MCP-1, MIP-1a, MIP-1b, RANTES, TNFa in mice serum were not affected by murine MSCs injection. Injection of CIMVs did not induce any statistically significant changes in cytokines level. Conclusion: Elevated levels of Eotaxin, G-CSF, IL-17A and IL-9 cytokines after the i.v.injection of murine MSCs suggest that moderate allergy inflammation was developed after the MSCs allotransplantation. CIMVs injection did not induced increase of cytokines level in mice serum indicating absence of immunogenicity. Taken together our results demonstrate that CIMVs show less/no immunogenicity compared to parental MSCs. We believe that small diameter, better biodistribution and fusion with host cells lead to the non-immunogenicity of CIMVs. Thus, CIMVs are confirmed to be a perspective, new biomimetic vector system. This study was funded by the grant of the President of the Russian Federation for state support of the leading scientific schools of the Russian Federation НШ-2603.2020.4. Kazan Federal University was supported by the Russian Government Program of Competitive Growth. Disclosures No relevant conflicts of interest to declare.

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Immunosuppressive properties of cytochalasin B-induced membrane vesicles of mesenchymal stem cells: comparing with extracellular vesicles derived from mesenchymal stem cells

Scientific Reports ◽

10.1038/s41598-020-67563-9 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

M. O. Gomzikova ◽

A. M. Aimaletdinov ◽

O. V. Bondar ◽

I. G. Starostina ◽

N. V. Gorshkova ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Extracellular Vesicles ◽

Cytochalasin B ◽

Membrane Vesicles ◽

Induced Membrane

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Angiogenic activity of cytochalasin B-induced membrane vesicles of human mesenchymal stem cells

10.1101/646398 ◽

2019 ◽

Author(s):

M.O. Gomzikova ◽

M.N. Zhuravleva ◽

V.V. Vorobev ◽

I.I. Salafutdinov ◽

A.V. Laikov ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cytochalasin B ◽

Human Mesenchymal Stem Cells ◽

Membrane Vesicles ◽

Molecular Composition ◽

Human Mscs ◽

Angiogenic Activity ◽

Induced Membrane

AbstractBackgroundThe cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown.ObjectivesThe objectives of this study were to analyzed the morphology, size distribution, molecular composition and angiogenic properties of CIMVs-MSCs.MethodsThe morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix.ResultsHuman CIMVs-MSCs retain parental MSCs content such as growth factors, cytokines, chemokines: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-α, TNF-β, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cellsin vitro. Finally, CIMVs-MSCs can induce angiogenesisin vivoafter subcutaneous injection into adult rats.ConclusionsHuman CIMVs-MSCs have similar content, immunophenotype and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases.

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Cytochalasin B-induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing TRAIL, PTEN and IFN-β1 Can Kill Carcinoma Cancer Cells

Tissue and Cell ◽

10.1016/j.tice.2021.101664 ◽

2021 ◽

pp. 101664

Author(s):

Daria S. Chulpanova ◽

Zarema E. Gilazieva ◽

Elvira R. Akhmetzyanova ◽

Sevindzh K. Kletukhina ◽

Albert A. Rizvanov ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cancer Cells ◽

Cytochalasin B ◽

Human Mesenchymal Stem Cells ◽

Membrane Vesicles ◽

Induced Membrane

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Intrapulmonary delivery of human umbilical cord mesenchymal stem cells attenuates acute lung injury by expanding CD4+CD25+forkhead boxp3 (FOXP3)+regulatory T cells and balancing anti- and pro-inflammatory factors

Klinische Pädiatrie ◽

10.1055/s-0032-1330787 ◽

2012 ◽

Vol 224 (07) ◽

Author(s):

R Wu ◽

J Sun ◽

ZC Han

Keyword(s):

Stem Cells ◽

T Cells ◽

Mesenchymal Stem Cells ◽

Acute Lung Injury ◽

Regulatory T Cells ◽

Lung Injury ◽

Umbilical Cord ◽

Inflammatory Factors ◽

Human Umbilical Cord

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Electrospun fibers enhanced the paracrine signaling of mesenchymal stem cells for cartilage regeneration

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02137-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nurul Dinah Kadir ◽

Zheng Yang ◽

Afizah Hassan ◽

Vinitha Denslin ◽

Eng Hin Lee

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Electrospun Fiber ◽

Cartilage Regeneration ◽

Conditioned Media ◽

Biologically Active ◽

Paracrine Signaling ◽

Therapeutic Value ◽

A Cell ◽

Fiber Sheet

Abstract Background Secretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage. Methods Conditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated. Results We showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production. Conclusions This study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

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Mesenchymal stem cells alleviate experimental immune-mediated liver injury via chitinase 3-like protein 1-mediated T cell suppression

Cell Death and Disease ◽

10.1038/s41419-021-03524-y ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Qiuli Liu ◽

Xiaoyong Chen ◽

Chang Liu ◽

Lijie Pan ◽

Xinmei Kang ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Liver Injury ◽

T Cell Activation ◽

Gene Set Enrichment Analysis ◽

Human Umbilical Cord ◽

Con A ◽

Immune Mediated

AbstractLiver diseases with different pathogenesis share common pathways of immune-mediated injury. Chitinase-3-like protein 1 (CHI3L1) was induced in both acute and chronic liver injuries, and recent studies reported that it possesses an immunosuppressive ability. CHI3L1 was also expressed in mesenchymal stem cells (MSCs), thus we investigates the role of CHI3L1 in MSC-based therapy for immune-mediated liver injury here. We found that CHI3L1 was highly expressed in human umbilical cord MSCs (hUC-MSCs). Downregulating CHI3L1 mitigated the ability of hUC-MSCs to inhibit T cell activation, proliferation and inflammatory cytokine secretion in vitro. Using Concanavalin A (Con A)-induced liver injury mouse model, we found that silencing CHI3L1 significantly abrogated the hUC-MSCs-mediated alleviation of liver injury, accompanying by weakened suppressive effects on infiltration and activation of hepatic T cells, and secretion of pro-inflammatory cytokines. In addition, recombinant CHI3L1 (rCHI3L1) administration inhibited the proliferation and function of activated T cells, and alleviated the Con A-induced liver injury in mice. Mechanistically, gene set enrichment analysis showed that JAK/STAT signalling pathway was one of the most significantly enriched gene pathways in T cells co-cultured with hUC-MSCs with CHI3L1 knockdown, and further study revealed that CHI3L1 secreted by hUC-MSCs inhibited the STAT1/3 signalling in T cells by upregulating peroxisome proliferator-activated receptor δ (PPARδ). Collectively, our data showed that CHI3L1 was a novel MSC-secreted immunosuppressive factor and provided new insights into therapeutic treatment of immune-mediated liver injury.

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