The comparison of adipose-derived stromal cells (ADSCs) delivery method in a murine model of hindlimb ischemia.

BACKGROUND: Adipose-derived stromal cells (ADSCs) demonstrate ability to promote tissue healing and down-regulate excessive inflammation. ADSCs have been used to treat critical limb ischemia in preclinical and clinical trials, but still, there is little known about their optimal delivery strategy. To date, no direct analysis of different methods of ADSCs delivery has been performed in the hindlimb ischemia model. Therefore, in this study we focused on the therapeutic efficacy of different ADSCs delivery methods in a murine model of hindlimb ischemia. METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hindlimb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6. ADSCs were delivered directly into ischemic muscle, into the contralateral muscle or intravenously. 7 and 14 days after the surgery, the gastrocnemius and quadriceps muscles were collected for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: Our research revealed that muscle regeneration, angiogenesis, arteriogenesis and macrophage infiltration in murine model of hindlimb ischemia differ depending on ADSCs delivery method. We have demonstrated that intramuscular method (directly into ischemic limb) of ADSCs delivery is more efficient in functional recovery after critical limb ischemia than intravenous or contralateral route. CONCLUSIONS: We have noticed that injection of ADSCs directly into ischemic limb is the optimal delivery strategy because it increases: (1) muscle fiber regeneration, (2) the number of capillaries and (3) the influx of macrophages F4/80+/CD206+.

Antitumor effect of anti-vascular therapy with STING agonist depends on the tumor microenvironment context.

Introduction: Targeting tumor vasculature is an efficient weapon to fight against cancer; however, activation of alternative pathways to rebuild the disrupted vasculature leads to rapid tumor regrowth. Immunotherapy that exploits host immune cells to elicit and sustain potent antitumor response has emerged as one of the most promising tools for cancer treatment, yet many treatments fail due to developed resistance mechanisms. Therefore, our aim was to examine whether combination of immunotherapy and anti-vascular treatment will succeed in poorly immunogenic, difficult-to-treat melanoma and triple-negative breast tumor models. Methods: Our study was performed on B16-F10 melanoma and 4T1 breast tumor murine models. Mice were treated with the stimulator of interferon genes (STING) pathway agonist (cGAMP) and vascular disrupting agent combretastatin A4 phosphate (CA4P). Tumor growth was monitored. The tumor microenvironment (TME) was comprehensively investigated using multiplex immunofluorescence and flow cytometry. We also examined if such designed therapy sensitizes investigated tumor models to an immune checkpoint inhibitor (anti-PD-1). Results: The use of STING agonist cGAMP as monotherapy was insufficient to effectively inhibit tumor growth due to low levels of STING protein in 4T1 tumors. However, when additionally combined with an anti-vascular agent, a significant therapeutic effect was obtained. In this model, the obtained effect was related to the TME polarization and the stimulation of the innate immune response, especially activation of NK cells. Combination therapy was unable to activate CD8+ T cells. Due to the lack of PD-1 upregulation, no improved therapeutic effect was observed when additionally combined with the anti-PD-1 inhibitor. In B16-F10 tumors, highly abundant in STING protein, cGAMP as monotherapy was sufficient to induce potent antitumor response. In this model, the therapeutic effect was due to the infiltration of the TME with activated NK cells. cGAMP also caused the infiltration of CD8+PD-1+ T cells into the TME; hence, additional benefits of using the PD-1 inhibitor were observed. Conclusion: The study provides preclinical evidence for a great influence of the TME on the outcome of applied therapy, including immune cell contribution and ICI responsiveness. We pointed the need of careful TME screening prior to antitumor treatments to achieve satisfactory results.

Mesenchymal stromal cells as carriers of IL-12 reduce primary and metastatic tumors of murine melanoma.

Due to immunosuppressive properties and confirmed tropism towards cancer cells mesenchymal stromal cells (MSC) have been used in many trials. In our study we used these cells as carriers of IL-12 in the treatment of mice with primary and metastatic B16-F10 melanomas. IL-12 has confirmed anti-cancer activity, induces a strong immune response against cancer cells and acts as an anti-angiogenic agent. A major limitation of the use of IL-12 in therapy is its systemic toxicity. The aim of the work was to develop a system in which cytokine may be administered intravenously without toxic side effects. In this study MSC were used as carriers of the IL-12. We confirmed antitumor effectiveness of the cells secreting IL-12 (MSC/IL-12) in primary and metastatic murine melanoma models. We observed inhibition of tumor growth and a significant reduction in the number of metastases in mice after MSC/IL-12 administration. MSC/IL-12 decreased vascular density and increased the number of anticancer M1 macrophages and CD8+ cytotoxic T lymphocytes in tumors of treated mice. To summarize, we showed that MSC are an effective, safe carrier of IL-12 cytokine. Administered systemically they exert therapeutic properties of IL-12 cytokine without toxicity. Therapeutic effect may be a result of pleiotropic (proinflammatory and anti-angiogenic) properties of IL-12 released by modified MSC.

The Proper Administration Sequence of Radiotherapy and Anti-Vascular Agent-DMXAA Is Essential to Inhibit the Growth of Melanoma Tumors.

Vascular disrupting agents (VDAs), such as DMXAA, effectively destroy tumor blood vessels and cause the formation of large areas of necrosis in the central parts of the tumors. However, the use of VDAs is associated with hypoxia activation and residues of rim cells on the edge of the tumor that are responsible for tumor regrowth. The aim of the study was to combine DMXAA with radiotherapy (brachytherapy) and find the appropriate administration sequence to obtain the maximum synergistic therapeutic effect. We show that the combination in which tumors were irradiated prior to VDAs administration is more effective in murine melanoma growth inhibition than in either of the agents individually or in reverse combination. For the first time, the significance of immune cells' activation in such a combination is demonstrated. The inhibition of tumor growth is linked to the reduction of tumor blood vessels, the increased infiltration of CD8+ cytotoxic T lymphocytes and NK cells and the polarization of macrophages to the cytotoxic M1 phenotype. The reverse combination of therapeutic agents showed no therapeutic effect and even abolished the effect of DMXAA. The combination of brachytherapy and vascular disrupting agent effectively inhibits the growth of melanoma tumors but requires careful planning of the sequence of administration of the agents.

Vascular disrupting agents in cancer therapy.

Tumor blood vessel formation is a key process for tumor expansion. Tumor vessels are abnormal and differ from normal vessels in architecture and components. Besides oxygen and nutrients supply, the tumor vessels system, due to its abnormality, is responsible for: hypoxia formation, and metastatic routes. Tumor blood vessels can be a target of anti-cancer therapies. There are two types of therapies that target tumor vessels. The first one is the inhibition of the angiogenesis process. However, the inhibition is often ineffective because of alternative angiogenesis mechanism activation. The second type is a specific targeting of existing tumor blood vessels by vascular disruptive agents (VDAs). There are three groups of VDAs: microtubule destabilizing drugs, flavonoids with anti-vascular functions, and tumor vascular targeted drugs based on endothelial cell receptors. However, VDAs possess some limitations. They may be cardiotoxic and their application in therapy may leave viable residual, so called, rim cells on the edge of the tumor. However, it seems that a well-designed combination of VDAs with other anti-cancer drugs may bring a significant therapeutic effect. In this article, we describe three groups of vascular disruptive agents with their advantages and disadvantages. We mention VDAs clinical trials. Finally, we present the current possibilities of VDAs combination with other anti-cancer drugs.

Brachytherapy in a Single Dose of 10Gy as an "in situ" Vaccination.

Radiotherapy (RT) is one of the major methods of cancer treatment. RT destroys cancer cells, but also affects the tumor microenvironment (TME). The delicate balance between immunomodulation processes in TME is dependent, among other things, on a specific radiation dose. Despite many studies, the optimal dose has not been clearly determined. Here, we demonstrate that brachytherapy (contact radiotherapy) inhibits melanoma tumor growth in a dose-dependent manner. Doses of 10Gy and 15Gy cause the most effective tumor growth inhibition compared to the control group. Brachytherapy, at a single dose of ≥ 5Gy, resulted in reduced tumor blood vessel density. Only a dose of 10Gy had the greatest impact on changes in the levels of tumor-infiltrating immune cells. It most effectively reduced the accumulation of protumorogenic M2 tumor-associated macrophages and increased the infiltration of cytotoxic CD8+ T lymphocytes. To summarize, more knowledge about the effects of irradiation doses in anticancer therapy is needed. It may help in the optimization of RT treatment. Our results indicate that a single dose of 10Gy leads to the development of a robust immune response. It seems that it is able to convert a tumor microenvironment into an "in situ" vaccine and lead to a significant inhibition of tumor growth.

Adipose tissue-derived stromal cells stimulated macrophages-endothelial cells interactions promote effective ischemic muscle neovascularization.

Neovascularization, the process of new blood vessels formation in response to hypoxia induced signals, is an essential step during wound healing or ischemia repair. It follows as a cascade of consecutive events leading to new blood vessels formation and their subsequent remodeling to a mature and functional state, enabling tissue regeneration. Any disruption in consecutive stages of neovascularization can lead to chronic wounds or impairment of tissue repair. In the study we try to explain the biological basis of accelerated blood vessels formation in ischemic tissue after adipose tissue-derived stromal cells (ADSCs) administration. Experiments were performed on mouse models of hindlimb ischemia. We have evaluated the level of immune cells (neutrophils, macrophages) infiltration. The novelty of our work was the assessment of bone marrow-derived stem/progenitor cells (BMDCs) infiltration and their contribution to the neovascularization process in ischemic tissue. We have noticed that ADSCs regulated immune response and affected the kinetics and ratio of macrophages population infiltrating ischemic tissue. Our research revealed that ADSCs promoted changes in the morphology of infiltrating macrophages and their tight association with forming blood vessels. We assume that recruited macrophages may take over the role of pericytes and stabilize the new blood vessel or even differentiate into endothelial cells, which in consequence can accelerate vascular formation upon ADSCs administration. Our findings indicate that administration of ADSCs into ischemic muscle influence spatio-temporal distribution of infiltrating cells (macrophages, neutrophils and BMDCs), which are involved in each step of vascular formation, promoting effective ischemic tissue neovascularization.

Author Correction: Combination of anti-vascular agent - DMXAA and HIF-1α inhibitor - digoxin inhibits the growth of melanoma tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Monitoring of diffusion properties and transverse relaxation time of mouse ischaemic muscle after administration of human mesenchymal stromal cells derived from adipose tissue.

OBJECTIVES: Application of non-invasive imaging methods plays an important role in the assessment of cellular therapy effects in peripheral artery disease. The purpose of this work was to evaluate the kinetics of MRI-derived parameters characterizing ischaemic hindlimb muscle after administration of human mesenchymal stromal cells derived from adipose tissue (hADSC) in mice. MATERIALS AND METHODS: MRI experiments were performed on a 9.4T Bruker system. The measurement protocol included transverse relaxation time mapping and diffusion tensor imaging. The monitoring period encompassed 14 days after femoral artery ligation and subsequent cell administration. The effect of hADSC transplantation was compared with the effect of normal human dermal fibroblasts (NHDFs) and phosphate-buffered saline injection. RESULTS: The most significant differences between the hADSC group and the remaining ones were observed around day 3 after ischaemia induction (increased transverse relaxation time in the hADSC group in comparison with the control group) and around day 7 (increased transverse relaxation time and decreased third eigenvalue of the diffusion tensor in the hADSC group in comparison with the control and NHDF groups) at the site of hADSC injection. Histologically, it was associated with increased macrophage infiltration at days 3-7 and with the presence of small regenerating fibres in the ischaemic tissue at day 7. CONCLUSIONS: Our results underscore the important role of macrophages in mediating the therapeutic effects of hADSCs and confirm the huge potential of magnetic resonance imaging in monitoring of cellular therapy effects.

The effect of culture media on large-scale expansion and characteristic of adipose tissue-derived mesenchymal stromal cells.

BACKGROUND: Adipose tissue-derived mesenchymal stromal cells (ASCs) have been shown to exhibit some promising properties of their use in regenerative medicine as advanced therapy medicinal products (ATMP). However, different sources of their origin, methods of isolation, and expansion procedures cause the laboratory and clinical results difficult to compare. METHODS: ASCs were isolated from lipoaspirates and cultured in three different medium formulations: αMEM and DMEM as a basal medium supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF as a gold standard medium. Subsequently, the impact of culture media on ASCs growth kinetics, their morphology and immunophenotype, ability to differentiate, clonogenic potential, and secretion profile was evaluated. RESULTS: All cultured ASCs lines showed similar morphology and similar clonogenic potential and have the ability to differentiate into three lines: adipocytes, osteoblasts, and chondroblasts. The immunophenotype of all cultured ASCs was consistent with the guidelines of the International Society for Cell Therapy (ISCT) allowing to define cells as mesenchymal stromal cell (MSC) (≥ 95% CD105, CD73, CD90 and ≤ 2% CD45, CD34, CD14, CD19, HLA-DR). The immunophenotype stabilized after the second passage and did not differ between ASCs cultured in different conditions. The exception was the ASCs grown in the presence of FBS and bFGF, which expressed CD146 antigens. The secretion profile of ASCs cultured in different media was similar. The main secreted cytokine was IL-6, and its level was donor-specific. However, we observed a strong influence of the medium formulation on ASCs growth kinetics. The proliferation rate of ASCs in medium supplemented with hPL was the highest. CONCLUSIONS: Culture media that do not contain animal-derived antigens (xeno-free) can be used to culture cells defined as MSC. Xeno-free medium is a safe alternative for the production of clinical-grade MSC as an advanced therapy medicinal product. Additionally, in such culture conditions, MSC can be easily expanded in accordance with the Good Manufacturing Process (GMP) requirements to a desired amount of cells for clinical applications.

Tumor Microenvironment as A "Game Changer" in Cancer Radiotherapy.

Radiotherapy (RT), besides cancer cells, also affects the tumor microenvironment (TME): tumor blood vessels and cells of the immune system. It damages endothelial cells and causes radiation-induced inflammation. Damaged vessels inhibit the infiltration of CD8+ T lymphocytes into tumors, and immunosuppressive pathways are activated. They lead to the accumulation of radioresistant suppressor cells, including tumor-associated macrophages (TAMs) with the M2 phenotype, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). The area of tumor hypoxia increases. Hypoxia reduces oxygen-dependent DNA damage and weakens the anti-cancer RT effect. It activates the formation of new blood vessels and leads to cancer relapse after irradiation. Irradiation may also activate the immune response through immunogenic cell death induction. This leads to the "in situ" vaccination effect. In this article, we review how changes in the TME affect radiation-induced anticancer efficacy. There is a very delicate balance between the activation of the immune system and the immunosuppression induced by RT. The effects of RT doses on immune system reactions and also on tumor vascularization remain unclear. A better understanding of these interactions will contribute to the optimization of RT treatment, which may prevent the recurrence of cancer.

Human ADSC xenograft through IL-6 secretion activates M2 macrophages responsible for the repair of damaged muscle tissue.

BACKGROUND: Adipose-derived mesenchymal stromal cells (ADSCs) are multipotent stromal cells. The cells secrete a number of cytokines and growth factors and show immunoregulatory and proangiogenic properties. Their properties may be used to repair damaged tissues. The aim of our work is to explain the muscle damage repair mechanism with the utilization of the human adipose-derived mesenchymal stromal cells (hADSCs). METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hind limb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6NCrl and NOD SCID mice. The mice received PBS- (controls) or 1 × 106 hADSCs. One, 3, 7, 14 and 21 days after the surgery, we collected the gastrocnemius muscles for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: The retention time of hADSCs in the limb lasted about 14 days. In the mice receiving hADSCs, the improvement in the functionality of the damaged limb occurred faster than in the control mice. More new blood vessels were formed in the limbs of the mice receiving hADSCs than in limbs of the control mice. hADSCs also increased the infiltration of the macrophages with the M2 phenotype (7-AAD-/CD45+/F4/80+/CD206+) into the ischemic limbs. hADSCs introduced into the limb of mice secreted interleukin-6. This cytokine stimulates the emergence of the proangiogenic M2 macrophages, involved, among others, in the repair of a damaged tissue. Both macrophage depletion and IL-6 blockage suppressed the therapeutic effect of hADSCs. In the mice treated with hADSCs and liposomes with clodronate (macrophages depletion), the number of capillaries formed was lower than in the mice treated with hADSCs alone. Administration of hADSCs to the mice that received siltuximab (human IL-6 blocker) did not cause an influx of the M2 macrophages, and the number of capillaries formed was at the level of the control group, as in contrast to the mice that received only the hADSCs. CONCLUSIONS: The proposed mechanism for the repair of the damaged muscle using hADSCs is based on the activity of IL-6. In our opinion, the cytokine, secreted by the hADSCs, stimulates the M2 macrophages responsible for repairing damaged muscle and forming new blood vessels.

Transcriptomes of human mesenchymal cells isolated from the right ventricle and epicardial fat differ strikingly both directly after isolation and long-term culture.

AIMS: Mesenchymal stromal cells isolated from different tissues are claimed to demonstrate similar therapeutic potential and are often incorrectly named mesenchymal stem cells. However, through comparison of such cells is lacking. This study aimed to compare the transcriptome of mesenchymal cells of the same phenotype isolated from the heart muscle and epicardial fat of the same patient, before and after culture. METHODS AND RESULTS: Cells were isolated from biopsies of the right ventricle and epicardial fat collected from five patients (three men and two women, mean age 59.4 ± 2.6) who underwent heart transplantation due to ischaemic cardiomyopathy. In both tissues, immunophenotyping revealed three distinct populations: (i)CD31- CD45- CD90+ CD34+ CD146- , (ii) CD31- CD45- CD90+ CD34- CD146+ , and (iii) CD31- CD45- CD90- CD34- CD146+ , of which only the first one could be grown after sorting. Material for RNA-seq was collected from these cells before culture (250 cells) and at passage 6 (5000 cells). Transcriptomic analysis revealed that cells of the same phenotype (CD31- CD45- CD90+ CD34+ CD146- ) upon isolation preferentially clustered according to the tissue of origin, not to the patient from whom they were isolated. Genes up-regulated in the right ventricle-derived cells were related to muscle physiology while down-regulated genes included those encoding proteins with transmembrane signalling receptor activity. After six passages, heart-derived and fat-derived cells did not acquire similar transcriptome. Cells isolated from the right ventricle in comparison with their epicardial fat-derived counterparts demonstrated higher level of transcripts related, among others, to RNA processing and muscle development. The down-regulated genes were involved in the nucleosome assembly, DNA packaging and replication, and interleukin-7-mediated signalling pathway. Cells from epicardial fat demonstrated higher heterogeneity both before and after culture. Cell culture significantly changed gene expression profile within both tissues. CONCLUSIONS: This study is an essential indication that mesenchymal cells isolated from different tissues do not demonstrate similar properties. Phenotypic identification and ease of isolation cannot be considered as a criterion in any therapeutic utilization of such cells.

Bioresorbable filomicelles for targeted delivery of betulin derivative - In vitro study.

Filomicelles (worm-like micelles) possess high drug loading capacity and long circulation time in the bloodstream. A novel approach can be filomicelles with folic acid (FA) as a targeting moiety. Folate-drug delivery systems can target FA receptors (FAR) that are overexpressed in several human carcinomas, which can potentially maximize therapeutic efficacy while minimizing side effects. The aim of this study was to develop filomicelles from combination of poly(L-lactide)-Jeffamine-folic acid and poly(L-lactide)-poly(ethylene glycol) for delivery of betulin derivative. Phosphate derivative of betulin reveals high cytotoxicity against cancer cells, however its application is restricted due to poor solubility in water. Incorporation into hydrophobic core of micelles can effectively solubilize the drug. Three kinds of micelles were obtained with high drug loading capacity. Based on TEM analysis, the copolymers formed exclusively filomicelles or mixture of filomicelles and spherical micelles. All kinds of micelles provided release of betulin derivative for over 9 days and apart the very initial phase displayed similar release profile. The influence of PLA block on initial burst effect was revealed. The in vitro cytotoxicity of betulin derivative loaded micelles against FAR-positive HeLa cells was confirmed, which proves their usefulness for targeted delivery of cytostatic drug.

Combination of anti-vascular agent - DMXAA and HIF-1α inhibitor - digoxin inhibits the growth of melanoma tumors.

Vascular disrupting agents as DMXAA inhibit tumor growth only for a short period of time followed by rapid tumor regrowth. Among others, hypoxia and presence of transcription factor HIF-1α are responsible for tumors regrowth. The aim of our study was to investigate the inhibition of murine melanoma growth by combining two agents: anti-vascular - DMXAA and the HIF-1α inhibitor - digoxin and explaining the mechanism of action of this combination. After DMXAA treatment tumor size was reduced only for a limited time. After 7 days regrowth of tumors was observed and number of vessels was increased especially in tumor's peripheral areas. DMXAA also induced an influx of immune cells: macrophages, CD8+ cytotoxic lymphocytes, NK cells, CD4+ lymphocytes. Administration of digoxin alone inhibited the growth of tumors. Administration of both agents in the proper sequence significantly inhibited the regrowth of tumors better than either agents alone. Combination therapy reduced number of newly formed vessels. In tumors of mice treated with combination therapy, the number of macrophages M1, CD8+ cytotoxic lymphocytes, NK cells and to a lesser extent CD4+ cells was increased. The combination of anti-vascular agents with HIF-1α inhibitors appears to be an effective therapeutic option.

M1-like macrophages change tumor blood vessels and microenvironment in murine melanoma.

Tumor-associated macrophages (TAMs) play a significant role in at least two key processes underlying neoplastic progression: angiogenesis and immune surveillance. TAMs phenotypic changes play important role in tumor vessel abnormalization/ normalization. M2-like TAMs stimulate immunosuppression and formation of defective tumor blood vessels leading to tumor progression. In contrast M1-like TAMs trigger immune response and normalize irregular tumor vascular network which should sensitize cancer cells to chemo- and radiotherapy and lead to tumor growth regression. Here, we demonstrated that combination of endoglin-based DNA vaccine with interleukin 12 repolarizes TAMs from tumor growth-promoting M2-like phenotype to tumor growth-inhibiting M1-like phenotype. Combined therapy enhances tumor infiltration by CD4+, CD8+ lymphocytes and NK cells. Depletion of TAMs as well as CD8+ lymphocytes and NK cells, but not CD4+ lymphocytes, reduces the effect of combined therapy. Furthermore, combined therapy improves tumor vessel maturation, perfusion and reduces hypoxia. It caused that suboptimal doses of doxorubicin reduced the growth of tumors in mice treated with combined therapy. To summarize, combination of antiangiogenic drug and immunostimulatory agent repolarizes TAMs phenotype from M2-like (pro-tumor) into M1-like (anti-tumor) which affects the structure of tumor blood vessels, improves the effect of chemotherapy and leads to tumor growth regression.

Antitumor activity of opiorphin, sialorphin and their conjugates with a peptide klaklakklaklak.

This is the study on the effect of opiorphin, sialorphin and their analogs on antitumor activity. We demonstrated that conjugation of opiorphin and sialorphin with a proapoptotic, antimicrobial peptide klak (klaklakklaklak) led to compounds (opio-klak and sialo-klak) that were cytotoxic against cancer cells (LN18, PC3, A549, HCT116 and B10-F16) in the MTT test. The conjugated analogs were designed to increase the effectiveness of the peptide. The opio-klak derivative was the most effective in the in vitro assays and led to a decrease in viability of cancer cells over time as compared with that of untreated controls. In contrast, treatment with either the untargeted klak peptide or opiorphin as a negative control led to a negligible loss in viability. Antitumor effect of the opio-klak was also observed in vivo in murine melanoma tumor-bearing mice. Cessation of peptide administration resulted in tumor regrowth. Our results are seemingly valuable for the development of opiorphin analogs with potential clinical applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

AIMS: The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. METHODS AND RESULTS: MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. CONCLUSION: In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

Combined Tumor Cell-Based Vaccination and Interleukin-12 Gene Therapy Polarizes the Tumor Microenvironment in Mice.

Tumor progression depends on tumor milieu, which influences neovasculature formation and immunosuppression. Combining immunotherapy with antiangiogenic/antivascular therapy might be an effective therapeutic approach. The aim of our study was to elaborate an anticancer therapeutic strategy based on the induction of immune response which leads to polarization of tumor milieu. To achieve this, we developed a tumor cell-based vaccine. CAMEL peptide was used as a B16-F10 cell death-inducing agent. The lysates were used as a vaccine to immunize mice bearing B16-F10 melanoma tumors. To further improve the therapeutic effect of the vaccine, we combined it with interleukin (IL)-12 gene therapy. IL-12, a cytokine with antiangiogenic properties, activates nonspecific and specific immune responses. We observed that combined therapy is significantly more effective (as compared with monotherapies) in inhibiting tumor growth. Furthermore, the tested combination polarizes the tumor microenvironment, which results in a switch from a proangiogenic/immunosuppressive to an antiangiogenic/immunostimulatory one. The switch manifests itself as a decreased number of tumor blood vessels, increased levels of tumor-infiltrating CD4(+), CD8(+) and NK cells, as well as lower level of suppressor lymphocytes (Treg). Our results suggest that polarizing tumor milieu by such combined therapy does inhibit tumor growth and seems to be a promising therapeutic strategy.

Characteristic of c-Kit+ progenitor cells in explanted human hearts.

According to literature data, self-renewing, multipotent, and clonogenic cardiac c-Kit(+) progenitor cells occur within human myocardium. The aim of this study was to isolate and characterize c-Kit(+) progenitor cells from explanted human hearts. Experimental material was obtained from 19 adult and 7 pediatric patients. Successful isolation and culture was achieved for 95 samples (84.1%) derived from five different regions of the heart: right and left ventricles, atrium, intraventricular septum, and apex. The average percentage of c-Kit(+) cells, as assessed by FACS, ranged between 0.7 and 0.9%. In contrast to published data we do not observed statistically significant differences in the number of c-Kit(+) cells between disease-specific groups, parts of the heart or sexes. Nevertheless, c-Kit(+) cells were present in significant numbers (11-24%) in samples derived from three explanted pediatric hearts. c-Kit(+) cells were also positive for CD105 and a majority of them was positive for CD31 and CD34 (83.7 ± 8.6 and 75.7 ± 11.4%, respectively). Immunohistochemical analysis of the heart tissue revealed that most cells possessing the c-Kit antigen were also positive for tryptase, a specific mast cell marker. However, flow cytometry analysis has shown cultured c-Kit(+) cells to be negative for hematopoietic marker CD45 and mast cell marker CD33. Isolated c-Kit(+) cells display mesenchymal stem cell features and are thought to differentiate into endothelial cells.