Therapeutic Mesenchymal Stem Cells Research Articles

Hyperthermia for Targeting Cancer and Cancer Stem Cells: Insights from Novel Cellular and Clinical Approaches.

The Cellular Heat Shock Response and in particular heat shock protein activation are vital stress reactions observed in both healthy and cancer cells. Hyperthermia (HT) has been proposed for several years as an advancing non-invasive cancer therapy. It selectively targets cancer cells through mechanisms influenced by temperature and temperature variations. This article delves into the impact of HT on cancer cells, especially cancer stem cells (CSCs), essential contributors to cancer recurrence and metastasis. HT has shown promise in eliminating CSCs, sensitizing them to conventional treatments and modulating the tumor microenvironment. The exploration extends to mesenchymal stem cells (MSCs), which exhibit both pro-tumorigenic and anti-tumorigenic effects. HT's potential in recruiting therapeutic MSCs for targeted delivery of antitumoral agents is also discussed. Furthermore, the article introduces Brain Thermodynamics-guided Hyperthermia (BTGH) technology, a breakthrough in temperature control and modulation of heat transfer under different conditions. This non-invasive method leverages the brain-eyelid thermal tunnel (BTT) to monitor and regulate internal brain temperature. BTGH technology, with its precision and noninvasive continuous monitoring capabilities, is under clinical investigation for applications in neurological disorders and cancer. The innovative three-phase approach involves whole-body HT, targeted brain HT, and organ-specific HT. In conclusion, the exploration of localized or whole-body HT offers promising avenues for cancer, psychiatric and neurological diseases. The ongoing clinical investigations and potential applications underscore the significance of understanding and harnessing heat's responses to enhance human health.

Treatment of Rheumatoid Arthritis (RA) Using Mesenchymal Stem Cells (MSC)

Rheumatoid arthritis (RA), as an persistent autoimmune condition, it is known for causing persistent synovitis, gradual deterioration of cartilage and bone, ultimately resulting in disability and widespread inflammation. Until now, the onset and development mechanisms of RA remain elusive, although the new technology and drugs can certainly help patients to certain degree. However, about 30% RA patients is not responsible for targeting treatment or serious side effects, such as bone marrow inhibition, organs dysfunction, and inflection due to immune inhibition. Research data showed that therapeutic mesenchymal stem cells (MSCs), is a novel therapeutic strategy with safety and effective to inhibit inflammation and promote regeneration of bone and cartilage, especially for refractory RA. Here, the author summarized the development MSC-based treatment for RA models and patients, analysis the optimistic outlook and possible difficulties for clinical applications in future.

Osteoarthritis—The Role of Mesenchymal Stem Cells in Cartilage Regeneration

Osteoarthritis (OA) is a condition that can cause substantial pain, loss of joint function, and a decline in quality of life in patients. Numerous risk factors, including aging, genetics, and injury, have a role in the onset of OA, characterized by structural changes within the joints. Most therapeutic approaches focus on the symptoms and try to change or improve the structure of the joint tissues. Even so, no treatments have been able to stop or slow the progression of OA or give effective and long-lasting relief of symptoms. In the absence of disease-modifying drugs, regenerative medicine is being investigated as a possible treatment that can change the course of OA by changing the structure of damaged articular cartilage. In regenerative therapy for OA, mesenchymal stem cells (MSCs) have been the mainstay of translational investigations and clinical applications. In recent years, MSCs have been discovered to be an appropriate cell source for treating OA due to their ability to expand rapidly in culture, their nontumorigenic nature, and their ease of collection. MSCs’ anti-inflammatory and immunomodulatory capabilities may provide a more favorable local environment for the regeneration of injured articular cartilage, which was thought to be one of the reasons why they were seen as more suited for OA. In addition to bone marrow, MSCs have also been isolated from adipose tissue, synovium, umbilical cord, cord blood, dental pulp, placenta, periosteum, and skeletal muscle. Adipose tissue and bone marrow are two of the most essential tissues for therapeutic MSCs. Positive preclinical and clinical trial results have shown that, despite current limitations and risks, MSC-based therapy is becoming a promising approach to regenerative medicine in treating OA.

Enhancing Anticancer Efficacy of Chemotherapeutics Using Targeting Ligand-Functionalized Synthetic Antigen Receptor-Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been studied for their potential in facilitating tumor-targeted delivery of chemotherapeutics due to their tumor-homing characteristics. We hypothesized that targeting effectiveness of MSCs can be further enhanced by incorporating tumor-targeting ligands on MSC surfaces that will allow for enhanced arrest and binding within the tumor tissue. We utilized a unique strategy of modifying MSCs with synthetic antigen receptors (SARs), targeting specific antigens overexpressed on cancer cells. MSCs were surface-functionalized by first incorporating recombinant protein G (PG) on the surface, followed by binding of the targeting antibody to the PG handle. We functionalized MSCs with antibodies targeting a tyrosine kinase transmembrane receptor protein, epidermal growth factor receptor (EGFR), overexpressed in non-small-cell lung cancer (NSCLC). The efficacy of MSCs functionalized with anti-EGFR antibodies (cetuximab and D8) was determined in murine models of NSCLC. Cetuximab-functionalized MSCs demonstrated improved binding to EGFR protein and to EGFR overexpressing A549 lung adenocarcinoma cells. Further, cetuximab-functionalized MSCs loaded with paclitaxel nanoparticles were efficient in slowing orthotopic A549 tumor growth and improving the overall survival relative to that of other controls. Biodistribution studies revealed a six-fold higher retention of EGFR-targeted MSCs than non-targeted MSCs. Based on these results, we conclude that targeting ligand functionalization could be used to enhance the concentration of therapeutic MSC constructs at the tumor tissue and to achieve improved antitumor response.

Fenoldopam Mesylate Enhances the Survival of Mesenchymal Stem Cells Under Oxidative Stress and Increases the Therapeutic Function in Acute Kidney Injury.

Mesenchymal stem cells (MSCs) have gained interest as an alternative therapeutic option for renal diseases, including acute kidney injury (AKI). However, their use is often limited owing to low survival rates in vivo. Fenoldopam mesylate (FD) is a selective dopamine D1 receptor agonist with antioxidative and anti-apoptotic roles. Herein, we investigated whether FD can enhance the survival of MSCs undergoing oxidative stress in vitro. In addition, the therapeutic effect of MSCs and FD-treated MSCs (FD-MSCs) was compared in a mouse model of AKI induced by cisplatin. The survival of MSCs under oxidative stress was augmented by FD treatment. FD induced the phosphorylation of cAMP response element-binding protein and AKT, contributing to enhanced growth compared with untreated MSCs. The expression of nuclear factor erythroid-2-related factor 2 (NRF2) and heme oxygenase-1 was increased by FD treatment, and nuclear translocation of NRF2 was found exclusively in FD-MSCs. FD downregulated BAX expression, increased the mitochondrial membrane potential, reduced reactive oxygen species generation, and decreased the apoptotic death of MSCs induced by oxidative stress. Moreover, renal function and tubular injury were improved in FD-MSCs compared with non-treated MSCs. Furthermore, tubular injury, apoptosis, and macrophage infiltration, as well as the serum level of tumor necrosis factor-α were reduced, while tubular cell proliferation was markedly increased in FD-MSCs compared with MSCs. Our study demonstrated that FD increases the survivability of MSCs in an oxidative environment, and its use may be effective in preparing robust therapeutic MSCs.

NSP4 and ORF9b of SARS-CoV-2 Induce Pro-Inflammatory Mitochondrial DNA Release in Inner Membrane-Derived Vesicles.

Circulating cell-free mitochondrial DNA (cf-mtDNA) has been found in the plasma of severely ill COVID-19 patients and is now known as a strong predictor of mortality. However, the underlying mechanism of mtDNA release is unexplored. Here, we show a novel mechanism of SARS-CoV-2-mediated pro-inflammatory/pro-apoptotic mtDNA release and a rational therapeutic stem cell-based approach to mitigate these effects. We systematically screened the effects of 29 SARS-CoV-2 proteins on mitochondrial damage and cell death and found that NSP4 and ORF9b caused extensive mitochondrial structural changes, outer membrane macropore formation, and the release of inner membrane vesicles loaded with mtDNA. The macropore-forming ability of NSP4 was mediated through its interaction with BCL2 antagonist/killer (BAK), whereas ORF9b was found to inhibit the anti-apoptotic member of the BCL2 family protein myeloid cell leukemia-1 (MCL1) and induce inner membrane vesicle formation containing mtDNA. Knockdown of BAK and/or overexpression of MCL1 significantly reversed SARS-CoV-2-mediated mitochondrial damage. Therapeutically, we engineered human mesenchymal stem cells (MSCs) with a simultaneous knockdown of BAK and overexpression of MCL1 (MSCshBAK+MCL1) and named these cells IMAT-MSCs (intercellular mitochondrial transfer-assisted therapeutic MSCs). Upon co-culture with SARS-CoV-2-infected or NSP4/ORF9b-transduced airway epithelial cells, IMAT-MSCs displayed functional intercellular mitochondrial transfer (IMT) via tunneling nanotubes (TNTs). The mitochondrial donation by IMAT-MSCs attenuated the pro-inflammatory and pro-apoptotic mtDNA release from co-cultured epithelial cells. Our findings thus provide a new mechanistic basis for SARS-CoV-2-induced cell death and a novel therapeutic approach to engineering MSCs for the treatment of COVID-19.

Deep learning multi-organ segmentation for whole mouse cryo-images including a comparison of 2D and 3D deep networks

Cryo-imaging provided 3D whole-mouse microscopic color anatomy and fluorescence images that enables biotechnology applications (e.g., stem cells and metastatic cancer). In this report, we compared three methods of organ segmentation: 2D U-Net with 2D-slices and 3D U-Net with either 3D-whole-mouse or 3D-patches. We evaluated the brain, thymus, lung, heart, liver, stomach, spleen, left and right kidney, and bladder. Training with 63 mice, 2D-slices had the best performance, with median Dice scores of > 0.9 and median Hausdorff distances of < 1.2 mm in eightfold cross validation for all organs, except bladder, which is a problem organ due to variable filling and poor contrast. Results were comparable to those for a second analyst on the same data. Regression analyses were performed to fit learning curves, which showed that 2D-slices can succeed with fewer samples. Review and editing of 2D-slices segmentation results reduced human operator time from ~ 2-h to ~ 25-min, with reduced inter-observer variability. As demonstrations, we used organ segmentation to evaluate size changes in liver disease and to quantify the distribution of therapeutic mesenchymal stem cells in organs. With a 48-GB GPU, we determined that extra GPU RAM improved the performance of 3D deep learning because we could train at a higher resolution.

Roe-inspired stem cell microcapsules for inflammatory bowel disease treatment

Mesenchymal stem cells (MSCs), which exert regulatory effects on various immune cells, have been a promising therapy for inflammatory bowel disease treatment. However, their therapeutic effects are limited by lack of nutritional supply, immune system attack, and low accumulation on the target site. Here, inspired by the natural incubation mechanism of roe, we present immune-isolating, wet-adhesive, and nutrient-rich microcapsules for therapeutic MSCs encapsulation. The adhesive shells were fabricated by ionic cross-linking of alginate and visible curing of epsilon-poly-L-lysine-graft-methacrylamide and dopamine methacrylamide, which encapsulated the liquid core of the MSCs and roe proteins. Due to the core-shell construction of the resultant microcapsules, the MSCs might escape from attack of the immune system while still maintaining immunomodulating functions. In addition, the roe proteins encapsulated in the core phase offered sufficient nutrient supply for MSCs' survival and proliferation. Furthermore, after intraperitoneal transplantation, the wet-adhesive radicals on the shell surface could immobilize the MSCs-encapsulating microcapsules onto the bowel. Based on these features, practical values of the roe-inspired microcapsules with MSCs encapsulation were demonstrated by applying them to treat dextran sulfate sodium (DSS)-induced colitis through increasing residence time, regulating immune imbalance, and relieving disease progression. We believe that the proposed roe-inspired microcapsules with MSCs encapsulation are potential for clinical application.

TRAIL-Armed ER Nanosomes Induce Drastically Enhanced Apoptosis in Resistant Tumor in Combination with the Antagonist of IAPs (AZD5582).

Although mesenchymal stem cells (MSCs) can be engineered to deliver the TNF-related apoptosis-inducing ligand (TRAIL) as an effective anticancer therapy, the clinical application is hampered by the costly manufacturing of therapeutic MSCs. Therefore, it is needed to find an alternative cell-free therapy. In this study, TRAIL-armed endoplasmic reticulum (ER)-derived nanosomes (ERN-T) are successfully prepared with an average size of 70.6nm in diameter from TRAIL transduced MSCs. It is demonstrated that the ERN-T is significantly more efficient for cancer cell killing than the soluble recombinant TRAIL (rTRAIL). AZD5582 is an antagonist of the inhibitors of apoptosis proteins (IAPs), and its combination with ERN-T induces strikingly enhanced apoptosis in cancerous but not normal cells. AZD5582 sensitizes resistant cancer cells to TRAIL through concomitant downregulation of IAP members like XIAP and the Bcl2 family member Mcl-1. Intravenously infused ERN-Ts accumulate in tumors for over 48 h indicating good tumor tropism and retention. The combination of ERN-T and AZD5582 drastically promotes therapeutic efficacy comparing with the cotreatment by rTRAIL and AZD5582 in a subcutaneous MDA-MB-231 xenograft tumor model. The data thus demonstrate that ERN-T can be a novel cell-free alternative to TRAIL-expressing MSC-based anticancer therapy and its efficacy can be drastically enhanced through combination with AZD5582.

Mesenchymal stem cells versus their extracellular vesicles in treatment of liver fibrosis: Is it possible to compare?

Liver fibrosis (LF) is a worldwide health problem that is associated with a range of complications and high mortality. Due to the scarcity of liver donors, mesenchymal stem cell (MSC) therapy emerged as an alternative therapeutic strategy. However, it is widely accepted that most of the transplanted MSCs exhibit their therapeutic impact mainly via a bystander paracrine (medicinal) capacity. In addition to their secretory proteins, MSCs also produce various types of extracellular vesicles (EVs) that are classified into three main subtypes: microvesicles, exosomes and apoptotic bodies. Thanks to their peculiar cargo composition (e.g., proteins, lipids, and nucleic acids), EVs serve as an advantageous candidate for cell-free therapy. Recently, MSC-derived EVs (MSC-EVs) have gained the podium due to their regenerative and immunomodulatory effect. In mitigation/treatment of LF, a plethora of recent studies have shown the anti-inflammatory, anti-fibrotic and cytoprotective effects of both MSCs and MSC-EVs in various in vitro and in vivo models of LF. However, despite the limited evidence, we sought in this mini review to sort out the established data and formulate several challenging questions that must be answered to pave the way for further clinical applications. One of the major questions to ask is “Which is the best therapeutic approach, MSCs or MSC-EVs?” We tried to highlight how difficult it might be to compare the two approaches while our understanding of both candidates is still deficient. Among the major obstacles against such comparison is the inaccurate equivalent dose determination, the unknown in vivo behavior, and the undetermined lifespan/fate of each. Currently, the fields of MSCs and MSC-EVs seem to be rich in ideas but lacking in appropriate technologies to test these ideas. Nevertheless, continuous efforts are likely to help resolve some of the challenges listed here.

A highly efficient non-viral process for programming mesenchymal stem cells for gene directed enzyme prodrug cancer therapy

Mesenchymal stem cells (MSCs) driven gene-directed enzyme prodrug therapy has emerged as a potential strategy for cancer treatment. The tumour-nesting properties of MSCs enable these vehicles to target tumours and metastases with effective therapies. A crucial step in engineering MSCs is the delivery of genetic material with low toxicity and high efficiency. Due to the low efficiency of current transfection methods, viral vectors are used widely to modify MSCs in preclinical and clinical studies. We show, for the first time, the high transfection efficiency (> 80%) of human adipose tissue derived-MSCs (AT-MSCs) using a cost-effective and off-the-shelf Polyethylenimine, in the presence of histone deacetylase 6 inhibitor and fusogenic lipids. Notably, the phenotypes of MSCs remained unchanged post-modification. AT-MSCs engineered with a fused transgene, yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) displayed potent cytotoxic effects against breast, glioma, gastric cancer cells in vitro. The efficiency of eliminating gastric cell lines were effective even when using 7-day post-transfected AT-MSCs, indicative of the sustained expression and function of the therapeutic gene. In addition, significant inhibition of temozolomide resistant glioma tumour growth in vivo was observed with a single dose of therapeutic MSC. This study demonstrated an efficient non-viral modification process for MSC-based prodrug therapy.

Efficacy of 3D Culture Priming is Maintained in Human Mesenchymal Stem Cells after Extensive Expansion of the Cells.

The use of non-optimal preparations of mesenchymal stem cells (MSCs), such as extensively expanded cells, might be necessary to obtain the large numbers of cells needed for many clinical applications. We previously demonstrated that minimally expanded (early passage) MSCs can be pre-activated as spheroids to produce potentially therapeutic factors in 3D cultures. Here, we used extensively expanded (late passage) MSCs and studied their 3D-culture activation potential. MSCs were culture-expanded as 2D monolayers, and cells from various passages were activated by 3D culture in hanging drops with either fetal bovine serum (FBS)-containing media or a more clinically-applicable animal product-free (xeno-free) media. Gene expression analyses demonstrated that MSC spheroids prepared from passage 3, 5, and 7 cells were similar to each other but different from 2D MSCs. Furthermore, the expression of notable anti-inflammatory/immune-modulatory factors cyclooxygenase-2 (PTGS2), TNF alpha induced protein 6 (TNFAIP6), and stanniocalcin 1 (STC-1) were up-regulated in all spheroid preparations. This was confirmed by the detection of secreted prostaglandin E2 (PGE-2), tumor necrosis factor-stimulated gene 6 (TSG-6, and STC-1. This study demonstrated that extensively expanded MSCs can be activated in 3D culture through spheroid formation in both FBS-containing and xeno-free media. This work highlights the possibility of activating otherwise less useable MSC preparations through 3D culture generating large numbers of potentially therapeutic MSCs.

Graft-versus-host disease suppression by mesenchymal stem cells as determined by cryo-imaging

Background & Aim GVHD limits success of allogeneic bone marrow transplantation (alloBMT). Mesenchymal stem cells (MSCs) has been shown to alleviate GVHD, but the biodistribution of the exogenous stem cells and mechanisms behind their protective effects remain to be fully elucidated. We hypothesize that allogeneic T-cells proliferate in secondary lymphoid organs (SLOs) and that therapeutic MSCs home there and suppress T-cell proliferation. Methods, Results & Conclusion We used multi-spectral mouse cryo-imaging (CryoVizTM) to quantitatively evaluate T-cell proliferation, MSC homing, and putative suppression of T-cells by MSCs. Cryo-imaging enables assessments of stem cell biodistribution, homing, and engraftment in whole-mouse with single-cell sensitivity. Briefly, a frozen mouse is sectioned and imaged in a tiled, automated fashion, providing ana¬tomical brightfield and molecular fluorescence, 3D mi-croscopic imaging, to detect labeled cells. Machine learning software accurately detects fluorescent clusters, enabling counting of injected cells. There were four groups of mice: alloBMT (with GVHD), syngeneic BMT (synBME without GVHD), alloBMT+MSCs (GVHD with MSC treatment), and synBMT+MSCs. T-cells were labeled with green-fluorescent CFSE dye, and MSCs were labeled with red-fluorescent quantum dots and tail vein injected. Cryo-imaging was performed on days 3, 4 and 5 post BMT. We examined the bio-distribution of MSCs and T-cells (Fig-A). MSCs homed to SLOs and localized in spleen marginal zone where they could come in contact with T-cells (Fig-B). When cells divide, CFSE dye is distributed to daughter cells and cell signal intensity decreases. Hence reduced CFSE signal intensity in the alloBMT group as compared to the synBMT group is consistent with a greater proliferation of T-cells as a precursor to GVHD (Figs-C-and-D). With MSCs, CFSE signal remained high, indicating a reduction in T-cell proliferation (Fig-E). Similar results were obtained in lymph nodes. Quantitative assays showed homing of about 3,000-4,000 MSCs in alloBMT, significantly more than in synBMT. There was a significant reduction in T-cell proliferation and SLO volumes when the alloBMT group was treated with MSCs. Treatment gave results between alloBMT (with GVHD) and syngeneic BMT (without GVHD). In conclusion, multispectral cryo-imaging allowed us to determine that only a few thousand MSCs could significantly affect T-cell proliferation in SLOs. This is likely one mechanism for MSC suppression of GVHD.

Mesenchymal stem cells targeting PI3K/AKT pathway in leukemic model.

Mesenchymal stem cells have therapeutic properties that are related to their potentials for trans-differentiation, immunomodulation, anti-inflammatory, inhibitory effect on tumor proliferation, and induction of apoptosis. This study was performed to analyze the role of mesenchymal stem cells as an alternative for cellular signaling growth factors involved in the pathogenesis of leukemogenesis in rats. Treatment of rats with 7,12-dimethyl benz [a] anthracene induced leukemogenesis appeared as a significant decrease in hematological parameters with concomitant significant increase in bone marrow oxidative and inflammatory indices (transforming growth factor beta and interleukin-6) in comparison with normal groups. On the contrary, Western immunoblotting showed a significant increase in the signaling growth factors: PI3K, AKT, mTOR proteins and a significant decrease in PTEN in 7,12-dimethyl benz [a] anthracene-treated group. In addition, a significant increase in the transcript levels of B cell lymphoma-2 protein gene in the 7,12-dimethyl benz [a] anthracene group, while that of C-X-C motif chemokine receptor-4 and B cell lymphoma-2 protein associated x-protein were significantly downregulated compared to controls. Meanwhile, therapeutic mesenchymal stem cells treatment predict a significant improvement versus 7,12-dimethyl benz [a] anthracene group through the modulation of growth factors that confront bone marrow dysplasia. In the same direction treatment of 7,12-dimethyl benz [a] anthracene group with mesenchymal stem cells, it induced apoptosis and increased the homing efficacy to bone marrow. In conclusion, mesenchymal stem cells improve hematopoiesis and alleviate inflammation, and modulated PI3K/AKT signaling pathway contributed to experimental leukemogenesis.

Encapsulated human mesenchymal stem cells (eMSCs) as a novel anti-cancer agent targeting breast cancer stem cells: Development of 3D primed therapeutic MSCs.

Breast cancer is a leading cause of mortality in women across the globe. The major reason for its recurrence and high mortality is due to the presence of a drug refractory and self-renewing population of cells, the cancer stem cells (CSCs). Mesenchymal stem cells (MSCs) have recently emerged as a promising cell-based therapeutic agent for the treatment of different cancer types. However, the anti-tumor effect of MSCs has been indicated to be substantially reduced by their in vivo tumor-trophic migration property and direct cell-to-cell integration with tumor stromal elements. To address this drawback, the present study uses a biomaterial, sodium alginate, for the encapsulation of MSCs from the perinatal tissue, Wharton's jelly (WJMSCs) into microbeads, to study the effect of WJMSCs beads on breast CSCs derived from two breast cancer cell lines, MDA-MB-231 and MCF7. Encapsulation with sodium alginate facilitated the prevention of direct cell-to-cell interaction and these microbeads provided a three-dimensional (3D) microenvironment for the encapsulated WJMSCs (eWJMSCs). Compared to two dimensional (2D) culture, eWJMSC increased proliferation of WJMSCs with an increase in pluripotency genes, epithelial to mesenchymal transition (EMT), immune-modulation, and angiogenesis. Interestingly, the tumor invasion suppressor protein E-cadherin was highly expressed in eWJMSCs as shown by Western blot analysis. In addition, eWJMSCs had an increased secretion of anti-inflammatory cytokines VEGF, TGF-β, TNF-α, IFN-γ, IL-10 and -6, and IL-3β when compared to 2D culture. Treatment of CSCs with eWJMSCs reduced cell viability, inhibited migration, and exerted an anti-angiogenic effect. eWJMSCs treatment of CSCs increased caspase 3/7 activity, nitric oxide production, and reactive oxygen species production, suggesting its anti-tumorigenic activity. Gene expression analysis revealed that CSCs treated with eWJMSCs had a downregulation of pro-proliferation markers, drug transporters, epithelial-mesenchymal transition-associated markers, and angiogenesis related genes. The mode of anti-proliferative action of WJMSCs beads was possibly through inhibition of the Wnt/β-catenin signaling pathway as indicated by the upregulation of the Wnt antagonists sFRP4 and DKK1. These data suggest that alginate-encapsulated WJMSCs could be an efficient cell contact-free system for developing stem cell-based therapies for CSCs.

Soluble matrix protein is a potent modulator of mesenchymal stem cell performance

We challenge the conventional designation of structural matrix proteins primarily as supporting scaffolds for resident cells. The extracellular matrix protein tropoelastin is classically regarded as a structural component that confers mechanical strength and resilience to tissues subject to repetitive elastic deformation. Here we describe how tropoelastin inherently induces a range of biological responses, even in cells not typically associated with elastic tissues and in a manner unexpected of typical substrate-dependent matrix proteins. We show that tropoelastin alone drives mesenchymal stem cell (MSC) proliferation and phenotypic maintenance, akin to the synergistic effects of potent growth factors such as insulin-like growth factor 1 and basic fibroblast growth factor. In addition, tropoelastin functionally surpasses these growth factors, as well as fibronectin, in allowing substantial media serum reduction without loss of proliferative potential. We further demonstrate that tropoelastin elicits strong mitogenic and cell-attractive responses, both as an immobilized substrate and as a soluble additive, via direct interactions with cell surface integrins αvβ3 and αvβ5. This duality of action converges the long-held mechanistic dichotomy between adhesive matrix proteins and soluble growth factors and uncovers the powerful, untapped potential of tropoelastin for clinical MSC expansion and therapeutic MSC recruitment. We propose that the potent, growth factor-like mitogenic and motogenic abilities of tropoelastin are biologically rooted in the need for rapid stem cell homing and proliferation during early development and/or wound repair.

Monocytic Cells Phagocytose Therapeutic Mesenchymal Stem Cells, which Induces Polarization, Relocation and Immune Regulation

Monocytic Cells Phagocytose Therapeutic Mesenchymal Stem Cells, which Induces Polarization, Relocation and Immune Regulation

Regulated Mesenchymal Stem Cells Mediated Colon Cancer Therapy Assessed by Reporter Gene Based Optical Imaging.

Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (p < 0.05 and 0.01) whereas no significant changes were observed in DOX(−) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(−) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.

The influence of hypoxia and IFN-γ on the proteome and metabolome of therapeutic mesenchymal stem cells

Over the past 15 years, mesenchymal stem cells (MSCs) have been assessed for their capacity to suppress inflammation and promote tissue repair. Regardless of whether the cells are primed (exposed to instructive cues) before administration, their phenotype will respond to environmental signals present in the pathophysiological setting being treated. Since hypoxia and inflammation coexist in the settings of acute injury and chronic disease we sought to explore how the proteome and metabolome of MSCs changes when cells were exposed to 48 h of 1% oxygen, interferon gamma (IFN-γ), or both cues together. We specifically focused on changes in cell metabolism, immune modulation, extracellular matrix secretion and modification, and survival capacity. IFN-γ promoted expression of anti-pathogenic proteins and induced MSCs to limit inflammation and fibrosis while promoting their own survival. Hypoxia instead led to cell adaptation to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. While dual priming resulted in additive effects, we also found many instances of synergy. These data lend insight to how MSCs may behave after administration to a patient and suggest how priming cells beforehand could improve their therapeutic capacity.

Feasibility of mesenchymal stem cell culture expansion for a phase I clinical trial in multiple sclerosis.

BackgroundMultiple sclerosis is an inflammatory, neurodegenerative disease of the central nervous system for which therapeutic mesenchymal stem cell transplantation is under study. Published experience of culture-expanding multiple sclerosis patients’ mesenchymal stem cells for clinical trials is limited.ObjectiveTo determine the feasibility of culture-expanding multiple sclerosis patients’ mesenchymal stem cells for clinical use.MethodsIn a phase I trial, autologous, bone marrow-derived mesenchymal stem cells were isolated from 25 trial participants with multiple sclerosis and eight matched controls, and culture-expanded to a target single dose of 1–2 × 106 cells/kg. Viability, cell product identity and sterility were assessed prior to infusion. Cytogenetic stability was assessed by single nucleotide polymorphism analysis of mesenchymal stem cells from 18 multiple sclerosis patients and five controls.ResultsOne patient failed screening. Mesenchymal stem cell culture expansion was successful for 24 of 25 multiple sclerosis patients and six of eight controls. The target dose was achieved in 16–62 days, requiring two to three cell passages. Growth rate and culture success did not correlate with demographic or multiple sclerosis disease characteristics. Cytogenetic studies identified changes on one chromosome of one control (4.3%) after extended time in culture.ConclusionCulture expansion of mesenchymal stem cells from multiple sclerosis patients as donors is feasible. However, culture time should be minimized for cell products designated for therapeutic administration.