STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY

STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Konference se koná ve spolupráci s Českou společností pro genovou a buněčnou terapii 14. listopadu 2012 Hotel Sladovna, Černá Hora

SBORNÍK ABSTRAKT

ISBN 978-80-7392-205-4

SBORNÍK ABSTRAKT Obsah Mesenchymal stromal cell-based suicide gene therapy of rat glioblastoma - preclinical study......... 4 Efficiency of Gene Therapy Mediated by MSC on Chemoresistant Colon Cancer Cells.......................... 5 Vplyv terapeutickej angiogenézy u pacientov s kritickou ischémiou dolných končatín na aktivitu trombocytov................................................................................................................................................ 6 ABC B5 expression induced by UV radiation in melanoma cells..................................................................... 7 Bladder cancer stromal fibroblasts can provide a niche for bladder carcinoma stem cells.................. 8 Mesenchymal stem cells as a relevant therapeutic tool in misfolded truncated tau induced neurodegeneration......................................................................................................................................................... 9 Pharmocological reversion of chemoresistance in colon cancer stem cells.............................................10 The role of hypoxia in expression of MDR-associated ABC transporters in embryonic stem cells...11 Flow cytometry analysis of stem cells indicated for cell therapy of critical limb ischemia.................12 Influence of the level of CD133 expression on adhesion and proliferation of cancer stem-like cells..................................................................................................................................................................13 Activation of hepatocyte growth factor pathway by prolonged beta-adrenergic stimulation is associated with increased levels of resident cardiac stem cell marker MDR-1 in rat left ventricle...........................................................................................................................................14 Cultured myoblasts from patient with MELAS syndrome...............................................................................15 Derivation of cardiomyocytes from embryonic stem cells.............................................................................16 Mesenchymal stromal cells drive significant biological changes in human breast cancer cells.......17 Intratékální aplikace lidských mesenchymálních stromálních buněk z kostní dřeně zlepšuje motoriku, zvyšuje přežívání a chrání perineuronální sítě potkanů s amyotrofickou laterální sklerózou.........................................................................................................................18 Freezing of mesenchymal stem cells in different media..................................................................................19 Exprese MHC-I na buňkách vedlejší populace TRAMP-C2, myšího modelu karcinomu prostaty.....20

Abstrakta neprošla jazykovou kontrolou.



STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Mesenchymal stromal cell-based suicide gene therapy of rat glioblastoma preclinical study Cihova M.1, Altanerova V.2, Altaner C.1,2, Ondicova K.3,4, Mravec B.3,4 Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia1 St. Elisabeth Cancer Institute, Bratislava, Slovakia2 Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia3 Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia4 Glioblastoma represents one of the most challenging human cancers to treat. It is known for its aggressive infiltrative growth, high proliferative rate and due to the presence of cancer stem cells also for the resistance to chemo and radiotherapy. Despite current advances in the standard of care therapies, the prognosis for glioblastoma patients has shown very little improvement over decades. The average survival time remains less than a year. Therefore, there is a great effort to find a more specific and effective approach. Mesenchymal stromal cells (MSC) known for their exclusive tumor tropic properties represent a very attractive tool for delivering deadly payload directly to the tumor site. We have modified adipose tissue-derived MSC in order to express yeast cytosine deaminase::uracil phospohoribosyl­transferase (CDy::UPRT) suicide gene, which effectively converts the relatively nontoxic prodrug 5– fluorocytosine (5-FC) into an active drug 5fluorouracil (5-FU) at the tumor bed. Such therapeutic cells have been intracerebrally administered to rats, either as a mixture with C6 cells or several days after the C6 induced tumor has grown. Our preclinical data show that this approach is efficacious, with no adverse side effects and that it holds potential for future clinical trials. We have demonstrated that the survival of C6 bearing animals depends on the dose of therapeutic cells and on the repeated application of therapeutic cells. Not only intracerebral co-administration of C6 cells and therapeutic stem cells, but also delayed administration of therapeutic cells after the tumor has grown to a distinct size induced complete tumor regression in a significant number of animals. Genetically modified therapeutic stem cells maintained their tumor tropic properties when injected to a distant intracranial site and effectively inhibited glioblastoma growth after 5-FC therapy. We have also demonstrated that continuous intracerebro-ventricular delivery of 5-FC using osmotic pump reduced the dose of prodrug required for the same therapeutic effect when injected intraperitoneally. Our results support the arguments to begin clinical studies for treatment of high-grade brain tumors.



SBORNÍK ABSTRAKT Efficiency of Gene Therapy Mediated by MSC on Chemoresistant Colon Cancer Cells Ďuriníková E., Matúšková M. Ústav experimentálnej onkológie SAV Retrovirally transduced human adipose tissue-derived mesenchymal stromal cells (AT-MSC) with so-called suicide genes, which catalyze the formation of highly toxic metabolites following the application of a nontoxic prodrug, were validated as cellular vehicles for gene-directed enzyme prodrug therapy (GDEPT) in many pre-clinical models. Tumor cells that continue to drive the growth and spread of colon cancer, particularly after surgery and drug treatment, represent an important therapeutic target for this disease. We aimed to evaluate the efficiency of three therapeutic GDEPT approaches on 5-flourouracil-resistant cells derived from colorectal carcinoma (HT-29/GFP/5FUR), which are able to proliferate in clinically relevant plasma concentration of 5-FU. Expression of surface markers did not change, as showed by flow cytometry. Bystander effect mediated by therapeutic AT-MSC on tumor cells was evaluated by fluorimetric and luminescent assay. The GDEPT approaches confirmed less sensitivity of prepared resistant cells. Fusion yeast gene cytosine deaminase::uracil phosphoribosyl­transferase with prodrug 5-fluorocytosine was more effective in elimination of chemoresistant cells in direct coculture in comparison to the single cytosine deaminase. We demonstrated inefficiency of therapeutic approach Herpes simplex virus thymidine kinase-transduced MSC with prodrug ganciclovir also by the confirmation, that cell line is incapable of gap junctions` formation. In colon cancer, CD133 marker has recently been used to enrich for a subset of tumor cells with tumor-initiating capabilities, and was therefore suggested to mark colon cancer stem cells. Immunomagnetically separated subpopulation of CD133+ cells of colorectal carcinoma is being more resistant to treatment when compared with cells negative to this marker. Further experiments are needed to find out if CD133 is really of that importance to be considered as appropriate marker for tumor-initiating cells, thus staining chemoresistant cells causing relapse of the tumor. Financial support: VEGA 2/014610, Slovak cancer research foundation grant NVR1, the Slovak Research and Development Agency under the contract No. APVV-0230–11.



STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Vplyv terapeutickej angiogenézy u pacientov s kritickou ischémiou dolných končatín na aktivitu trombocytov Fedor M.1, Fedorová J.2, Hudeček J.1, Staško J.1, Chudý P.1, Šinák I.3, Talapková R.3, Kubisz P.1,2 Klinika hematológie a transfuziológie JLF UK a UN Martin1 Hemo Medika s.r.o, centrum hemostázy a trombózy, Martin2 Klinika cievnej chirurgie, JLFUK a UN Martin3

Cieľ Cieľom štúdie bolo zistiť a popísať rozdiely v imunofenotype trombocytov u pacientov s kritickou ischémiou dolných končatín (CLI, Critical Limb Ischemia) liečených terapeutickou angiogenézou (TA) s použitím mononukleárnych kmeňových buniek kostnej drene (BMMNC , Bone marrow-derived mononuclear cells) v porovnaní s kontrolnou skupinou zdravých pacientov za účelom zhodnotenia vplyvu terapeutickej angiogenézy na expresiu membránových markerov trombocytov.

Metodika Sledovaný súbor tvorili pacienti s kritickou ischémiou dolných končatín liečení terapeutickou angiogenézou (n=26) a kontrolná skupina zdravých pacientov (n=10) . Zo spina iliaca anterior posterior sme odobrali 650 ml kostnej drene. Získaný materiál bol centrifugovaný (22°C, 3100 rpm, 2 min) a následne boli systémom Optipress separované BMNC (100–120ml). Kmeňové bunky sme implantovali do ischemickej končatiny intramuskulárnou cestou. Po zákroku boli pacienti sledovaní v časovom horizonte 1,3 a 6 mesiacov. Na imunofenotypizáciu trombocytov sme využili prietokovú cytometriu , s použitím protilátok CD41FITC, CD61PE, CD51FITC, CD62PE, CD36FITC, CD63PE, CD29FITC, CD49bFITC.

Výsledky U pacientov s CLI bola zistená výrazne nižšia expresia CD41, CD61, CD51, CD63 a CD49b v porovnaní s kontrolnou skupinou. Expresia P-selektínu (CD62P) bola u pacientov s CLI zvýšená avšak v období 1 mesiaca po liečbe došlo k výraznému poklesu oproti bazálnej hodnote . Doštičkový glykoproteín GPIV (CD36) bol výrazne zvýšený v období pred liečbou a taktiež aj po uplynutí jedného mesiaca. Hladina β1-integrínu (CD29) vykazovala v období po liečbe progresívny pokles. Preukázali sme koreláciu medzi poklesom hladín CD29 a CD36.

Záver Trombocyty pacientov s kritickou ischémiou dolných končatín exprimujú P-selektín, GPIV a β1-integrín vo vyššej miere ako trombocyty zdravých pacientov. V obdobi po liečbe terapeutickou angiogenézou s použitím BMMNC sa expresia uvedených markerov znižuje. Príčinou je pravdepodobne zvýšená spotreba trombocytov počas angiogenézy.



SBORNÍK ABSTRAKT ABC B5 expression induced by UV radiation in melanoma cells Gudernová I., Procházková J. Přírodovědecká fakulta, Masarykova univerzita,Ústav experimentální biologie, Oddělení fyziologie a imunologie živočichů Malignant melanoma is a type of cancer that arises from melanocytes, specialized cells that produce the pigment melanin. Formation of melanin is generally called melanogenesis, which includes biogenesis of melanosomes, melanin synthesis and detoxification of endogenous cytotoxic agents through melanin. Treatment of melanoma is complicated because the cells are usually resistant to chemotherapy and radiation therapy. The reason for this resistance may be detoxification of drug through melanin synthesis or increased export of anticancer drugs through the cytoplasmic membrane. This efflux is mediated by family ATP-binding cassette B (ABC B) transporters, such as ABC B1 (MDR1, Pgp) and ABC B5. The expression of ABC B5 is increased on the surface of melanocytes, melanosomes, as well as in melanomas resistant to chemotherapeutics. ABC B5 is also increased in CD133+ progenitor melanocytes and melanoma cancer stem cells. Melanoma cells that lack ability to synthesize melanin showed absence ABC B5 transporter suggesting the involvement ABC B5 in melanogenesis. The aim of this project was to determine the role of ABC B5 transporter in melanogenesis induced by UV radiation. As model cell lines were used melanoma tumor lines: Mel4M (gift of prof. Polakowska, INSERM, Lille, France), A375, Mel-Juso and IPC298 (gift of Dr. Uldrian, LF, Masaryk University, Brno). Using immunocytoche­mistry, Western blotting and qRT-PCR we tested expression of the ABC B5. The next aim was to determine whether the induction of melanogenesis UV radiation leads to changes in the level of melanin and ABC B5 under the influence of PI3K/Akt, MAPKs, and STAT pathways. This project is supported by grants MUNI/C/0829/2011 and MUNI/A/0975/2009.



STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Bladder cancer stromal fibroblasts can provide a niche for bladder carcinoma stem cells Hatina J., Šrámek J., Tuková J., Fernandes M., Koutová L. Universita Karlova, lékařská fakulta v Plzni, ústav biologie According to the cancer stem cells hypothesis, tumours are organized in a hierarchical manner, with cancer stem cells (CSC) at the top of this intrinsic cellular hierarchy. Mounting evidence indicates that stromal cells contribute crucially to the regulation and maintenance of CSC during tumour development and progression. However, experimental systems to study this niche-providing activity of cancer stromal cells are relatively scarce. We have recently established a pair of carcinoma (BC44) and carcinoma-associated fibroblast (BC44Fibr) ? cell lines derived from the same urothelial tumour. BC44Fibr cells display typical attributes of carcinoma-associated fibroblasts (ubiquitous expression of Vimentin and Smooth Muscle α-Actin, prevalent expression of Fibroblast Activation Protein). Focal expression of CD13 suggests their bladder stroma origin, nevertheless, the cells could be successfully differentiated into osteoclasts and chondrocytes, a property of cells originating from tumour-recruited mesenchymal stem cells. Co-culture of BC44Fibr and bladder carcinoma cell lines (BC44, SW780, HT1197) revealed that carcinoma cells strongly positive for putative CSC markers (CK-17, 67 KDa Laminin Receptor, CD44v6) were located at the very margin of carcinoma cell colonies, i.e. in direct contact with co-cultured carcinoma fibroblasts. Also, coculture with BC44Fibr significantly enhanced the ancorage-independent growth of BC44, as well as of another poorly clonogenic bladder cancer cell line BC61. The stem cell promoting activity of BC44Fibr cells manifested even in co-culture with normal skin keratinocytes. We believe that this experimental system could be very valuable in deciphering the mechanisms involved in stem cell promoting activity of carcinoma fibroblasts. Supported by the by the specific student research grants of the Charles University SVV-2012– 264804 and SVV-2012–264806.



SBORNÍK ABSTRAKT Mesenchymal stem cells as a relevant therapeutic tool in misfolded truncated tau induced neurodegeneration Kazmerova Z.1,2, Zilka N.1,2, Zilkova M.1,2, Novak M.1,2 Institute of Neuroimmunology, Slovak Academy of Sciences, Centre of Excellence for Alzheimer´s Disease and Related Disorders, Dubravska cesta 9, 845 10 Bratislava, Slovak Republic1 AXON Neuroscience SE, Grӧsslingova 45, 811 09 Bratislava, Slovak Republic2 Neurofibrillary pathology, which is the major pathological hallmark of Alzheimer?s di­sease, is composed of truncated misordered tau and hyperphosphorylated forms of tau protein. To validate a pathological activity of truncated misordered forms of tau protein in vitro, we expressed the most active form of human truncated tau protein (tau151–391, 4R) in neuroblastoma cell line SHSY5Y. We demostrated that truncated tau slowed down cell proliferation, reduced the metabolic activity and induced specific caspase-3?independent apoptotic-like cell death?tauoptosis in our AD cell model. In last 20 years exploitation of stem cells and their neuroregenerative and immunomodulatory properties revealed that there is a huge possibility of their exploitation in cellmediated therapy at different neurodegenerative diseases. According these evidences, we aimed at the effect of mesenchymal stem cells and their secretome on misfolded truncated tau proteininduced cell death in our AD cell model. In the present study, misfolded truncated tau protein expressing cells after treatment with 10 μM all-trans retinoic acid were co-cultivated with MSCs or supplemented with MSC secretome for 6 and 9 days. We demonstrated that both MSCs and MSC secretome promoted survival and increased the metabolic activity of the cells. Moreover, cocultivation with stem cells induced cell differentiation and formation of neurites with numerous varicosities in our AD cell model. Strikingly, treatment had no effect on tau expression suggesting that MSC induced self-protecting mechanism that prevented misfolded truncated tau expressing cells from tauoptosis. Our results indicate that mesenchymal stem cells and their secretome are able to rescue the Alzheimer?s disease cell model from cell death induced by misfolded truncated tau and suggest that stem cells therapy may represent an alternative therapeutic avenue for treatment of human Alzheimer?s disease and related tauopathies. Acknowledgement: This work was supported by Axon Neuroscience and research grants VEGA 2/0144/08, VEGA 2/0067/10, VEGA 2/0193/11, LPP-0039–09, APVV 0631–07 and structural fund 26240220046, BIOMARKAPD (Biomarkers for Alzheimer?s disease and Parkinson?s di­sease, Joint Programming in Neurodegenerative Diseases).



STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Pharmocological reversion of chemoresistance in colon cancer stem cells Kozovská Z.1, Gábrišová V2., Ďuriníková E.1, Matúšková M.1, Kučerová L.1 Laboratory of Molecular Oncology, Cancer Research Institute of Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovak Republic1 Faculty of Pharmacy, Comenius University in Bratislava, Odbojárov 10, 832 32 Bratislava, Slovak Republic2 The population of tumour cells is heterogenic and consists of several subpopulations. Cancer stem cells represent chemoresistant subpopulation which is largely responsible for chemotherapy failure. We have derived chemo-resistant cells from human colorectal adenocarcinoma cell line HT-29/GFP by continuous cultivation in medium with gradually increasing concentration of 5-fluorouracil (5FU). We compared the sensitivity of parental HT-29/GFP and resistant cell line HT-29/GFP/5FUR to 5FU and Cisplatin (CisPt) alone and in the presence of compound diethylaminoben­zaldehyd (DEAB), a specific inhibitor of Aldehyde dehydrogenase 1A1. We demonstrated that DEAB sensitizes cells to chemotherapy by fluorimetric assay. The Activity of ALDH1A1 was proved by functional assay ALDEFLOUR. This assay confirmed the functional inhibition of aldehyde dehydrogenase 1A1 by DEAB in tested cells. We determined expression profile of the parental and resistant population of cancer cells by qPCR with expression array for Human drug transporters. Expression of cancer stem cells markers (ALDH1A1, CD133) and expression of enzymes involved in metabolism of 5-FU was markedly different in resistant cells. The compound DEAB can restore 5-FU sensitivity in both tested lines (parental and resistant). Next, we plan to knock down the expression of genes involved in chemoresistance by specific siRNA and based on previous results we will test the expression profile of such cells. We will also analyse expression of relevant genes in stem cells subpopulations (ALDH1+ and CD133+). Financial support: The Slovak Research and Development Agency under the contract No. APVV0230–11, Cancer Research foundation NVR 1, VEGA 2/0146/10

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SBORNÍK ABSTRAKT The role of hypoxia in expression of MDR-associated ABC transporters in embryonic stem cells Lánová M., Kučera J., Kotasová H., Procházková J., Pacherník J. Ústav experimentální biologie, Přírodovědecká fakulta, Masarykova univerzita High level of ATP binding cassette (ABC)transporter expression is a typical feature of stem cells and many types of cancer stem cells. Multidrug resistance (MDR) of cancer cells is mainly caused by overexpression of ABC B1/ P-glycoprotein/MDR1, ABC C1/multidrug resistance-associated protein 1 (MRP1), and ABC G2/BCRP (Breast cancer resistance protein)(1). These transporters protect stem cells against toxic substances of either endogenous or exogenous origin. Moreover, they also have an important role in regulating the differentiation and proliferation of diverse cell types. Current data suggest that distribution of oxygen and nutrients is also involved in regulation of differentiation. Experiments with cultivation of different types of stem cells in vitro confirm the positive role of hypoxia in stemness maintenance (2). Stabilization of hypoxia-inducible factor (HIF) by hypoxia results in activation of this transcription factor for genes adapting the metabolism of cells to the lack of oxygen and simultaneously triggers the expression of growth factors that induce angiogenesis. In our experiments, we confirmed the expression of MDR transporters in mES cells by qRT-PCR. The highest expression showed ABC G2, which was previously proved to protects stem cells from oxidative stress (3). Functional analysis by flow cytometry also demonstrated the hight efflux activity of this transporter. Exposure of mES cells to hypoxia for 24 h resulted in decreased expression of the studied ABC transporters. This decrease can be explained by induction of cell differentiation as a result of low oxygen concentrations. The absence of HIF1 alpha did not reduce level of mRNA expression of MDR transporters and also lack of p38 kinase (kinase sensitive to reactive oxygen species (ROS) ? the key regulator of cellular response to oxidative stress) did not affect expression of MDR proteins. (1) Stavrovskaya, A. A., Stromskaya, T. P. 2008: Biochemistry (Mosc), 73(5): 592–604. (2) Simon, M. C., Keith, B. 2008. Nat Rev Mol Cell Biol. 9(4): 285–296. (3) Susanto, J. et al. 2008. PLoS One 3(12): e4023. Supported by internal grant agency of Masaryk University ? the rector?s grant MUNI/C/0836/ 20­11.>

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STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Flow cytometry analysis of stem cells indicated for cell therapy of critical limb ischemia Mlynárová J.1, Musil P.1, Klepanec A.2, Maďarič J.2, Kyselovič J.1 Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic1 National Cardiovascular Institute, Bratislava, Slovak Republic2 Introduction: To date there are promising data showing a therapeutic effect of bone marrow cells application in patients with critical limb ischemia. The CD34+ progenitor cells are supposed to participate in the process of angiogenesis, however, the role of particular cell types in neoangiogenesis and regeneration is not clearly elucidated. Therefore the aim of our work was to analyse CD34+, CD133+ progenitor cell and CD14+ cell count in bone marrow aspirate and bone marrow cell concentrate. Methods: Bone marrow aspirate was collected from 5 patients with critical limb ischemia. Aspiration was performed under aseptic conditions under analgosedation from both posterior iliac crests. 240 ml of bone marrow aspirate was collected in a system containing ACD-A anticoagulant and processed by gradient density centrifugation (SmartPreP2 Bone Marrow Aspirate Concentrate System, Harvest, Plymouth, MA, USA). Flow cytometry was used to analyse CD34+, CD133+ CD14+ cells in obtained bone marrow cell concentrate and bone marrow aspirate. Results: From total 1.99×107 viable leukocytes per millilitre in bone marrow aspirate there was 0.28% of viable CD34+, 0.15% of viable CD133+ and 3% of viable CD14+ cells. From total 9.01×107 viable leukocytes per millilitre in bone marrow cell concentrate there was 0.34% of viable CD34+, 0.09% of viable CD133+ and 2.92% of viable CD14+ cells. Number of CD34+, CD133+, CD14+ cells and total number of leukocytes was 6.24, 3.55, 4.42 and 4.93 respectively times higher in bone marrow cell concentrate compared to bone marrow aspirate. Conclusion: **In our work we characterised bone marrow aspirate and bone marrow cell concentrate by analysing CD34+, CD133+ and CD14+ cell count. Knowing the cellular composition of bone marrow cell concentrate designated to cell therapy of critical limb ischemia could help in studying angiogenesis induction in ischemic tissue. **Acknowledgement: This work was supported by grants VEGA 1/0786/11, ITMS 26240220071

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SBORNÍK ABSTRAKT Influence of the level of CD133 expression on adhesion and proliferation of cancer stem-like cells Pernicová Z.1,2,3, Fedr R.1, Kozubík A.1,3, Souček K.1,2, Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic1 Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne´s University Hospital Brno, Brno, Czech Republic2 Department of Experimental Biology, Faculty of Sciences, Masaryk University,Brno, Czech Republic3 There are two main published approaches, how to detect prostate stem cells in mice using combination of several extracellular markers, as for example CD44, CD117, CD133 (Prominin-1), Trop-2. In prostate cancer, cells with phenotype similar to stem cells were identified, and recently, subpopulation of cells expressing some of these markers was also identified in several cell lines cultivated in vitro. Here we used mouse prostate cancer cell line cE2, which we found out to express selected cancer stem cell markers, namely CD133 and CD44. We focused on elucidating whether level of CD133 expression can affect several biological processes. We used clonogenic assay for elucidating the proliferative capacity of CD133low versus CD133high cells, and found out that CD133high cells have higher clonogenic capacity. Further, this correlated with increased proliferation of CD133high cells assessed using label-free real time cell analyzer xCELLigence. Finally, using this system, we examined, how expression of CD133 affects adhesion properties of CD133low and CD133high cells. Taken together, we found out that mouse prostate cancer cell line cE2 express cancer stem cell markers, and that the level of CD133 expression affects proliferation, clonogenic capacity, and adhesion of these cells. This work was supported by grants IGA MZD 9600–4/2008, IGA MZD NT1357–4/2012, and by project FNUSA-ICRC (no. CZ 1.05/1.1.00/02­.0123) from the European Regional Development Fund.

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STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Activation of hepatocyte growth factor pathway by prolonged beta-adrenergic stimulation is associated with increased levels of resident cardiac stem cell marker MDR-1 in rat left ventricle Pivackova L., Cernecka H., Turcekova K., Olvedy M., Ochodnicky P., Gajdacova B., Adameova A., Kyselovic J., Klimas J., Krenek P. Farmaceutická fakulta univerzity Komenského v Bratislave Aims: Hepatocyte growth factor (HGF) in addition to its angiogenic, antiapoptotic and antifibrotic effects also acts as potent mitogenic, motogenic and differentiation factor on cardiac and mesenchymal stem cells. We investigated HGF and MDR-1 (stem cell marker) expression during the development of isoproterenol-induced heart failure in rat left ventricles. Methods: Adult male Wistar rats (n=7 per group) were administered isoproterenol at dose 5 mg/kg i.p once a day. Rats were sacrificed after 1, 2, 4, 8 days of treatment. Total RNA was isolated from left ventricles, reversetranscribed and analyzed by real-time PCR analysis using gene-specific primers for hepatocyte growth factor (HGF), HGF receptor (c-met), hepatocyte growth factor activator inhibitor-1 and –2 (HAI-1, HAI-2), multidrug resistance protein-1 (MDR-1) and skeletal muscle alpha-actin (ACTA1) as a marker of cardiac hypertrophy. Statistical evaluation was performed by ANOVA. Results: 30 % mortality was observed within first 4 days of treatment, but no rats died later. Left ventricular mass and cardiac hypertrophy marker ACTA1 were increased from day 1 until the end of the experiment (P<0.05). HGF expression increased after 2 days of treatment and remained elevated until day 8 (P<0.05). c-met expression increased after 4 days (P<0.05). HAI-1 mRNA levels were elevated on day 1 and day 2 (P<0.05), then they returned to control levels. Expression of HAI-2 mRNA remained unchanged during the experiment. Expression of MDR-1 began to rise on day 2, peaked on day 4 and remained at this level until day 8 (P<0.05). Conclusions: Chronic isoproterenol administration in the rat is able to induce HGF production, however elevated levels of HAI-1 during first 2 days may abolish HGF activation. The HGF pathway could become fully functional after 4 days of ISO administration, when c-met is upregulated. This coincides with upregulation of MDR-1 as marker of one subpopulation of cardiac stem cells, which could together with antiapoptotic and angiogenic properties of HGF also contribute to observed decrease in mortality after 4 days of treatment. The project was supported by: VEGA 1/0981/12, 1/0357/09, 1/0377/09, 1/0707/09.

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SBORNÍK ABSTRAKT Cultured myoblasts from patient with MELAS syndrome Sladkova J., Docekalova D., Hansikova H., Honzik T., Tesarova M., Zeman J. Laboratoř pro studium mitochondriálních poruch To study the expression of pathogenic mtDNA mutations are available different cells types (fibroblasts, lymphocytes) but using primary myoblast cultures offer several important advantages: skeletal muscle fibers are postmitotic multinuclear syncytia that cannot grown in tissue culture. They are, however, surrounded by mononuclear, undifferentiated myoblasts called satellite cells that can proliferate in culture. Because satellite cells enter a phase of quiscence at the end of postnatal growth, it is likely that the turnover of mitochondria and mtDNA is extremely slow compared to that in differentiated muscle fibers. Thus it is likely that the relative proportions of mutant and wild type mtDNAs in the satellite cell population reflects the degree of heteroplasmy that existed in the myoblast precursor population during embryological development. A comparison with adult muscle showed the segregation of mtDNA subsequent to terminal differentiation. In our study we tested the level of heteroplasmy of mtDNA mutations A3234G and mitochondrial ultrastructure in primary myoblast culture derived from one patient with MELAS syndrome. The muscle satellite cells were isolated by explant technique, which uses the intrinsic property of satellite cells to become activated and migrate to a site of lesion.Pre-plating technique was used to ensure the maximum purity of myoblasts. Mitochondrial reticulum visualized by MitoTracker Red CMXRos was scarce in myoblast cells compared to fibroblasts. Megamitochondria were sporadically observed. Ultrastructural analysis of patient myoblasts revealed a heterogenous mixture of abnormal swelling mitochondria with unusual and sparse cristae. Molecular genetic analysis, applied for assessment of mtDNA heteroplasmy level of both myoblasts and skeletal muscle of the patient, revealed certain increase of the heteroplasmy degree of skeletal muscle. This variation suggests investigation of a possible correlation or relationship with pathologic morphology. Detailed biochemical and molecular genetic analysis of myoblasts of several patients with mitochondrial disorders is currently proceeding. Supported by IGA NT/11186–5/2010

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STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Derivation of cardiomyocytes from embryonic stem cells Sýkorová D., Procházková J., Binó L., Kotasová H., Kubala L., Pacherník J. Masarykova Univerzita Preparation of cardiomyocytes from pluripotent embryonic stem (ES) cells represents a strong in vitro tool for study of cardiomycyte development and functions. Such new cardiomyocytes may be potentially used later for cell therapy and tissue repair. In our work we first characterized cardiomyogenesis and improved the currently used differentiation protocol in ES cells. Using our protocol we were also able to prepare significantly enriched cardiomyocytes population by simple mechanical collection of beating cardiomyocytes. Secondly, with aim to further improve purity of differentiating cardiomyocytes, we prepared new ES cell line modified by vectors allowing selection of cardiomyocytes.

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SBORNÍK ABSTRAKT Mesenchymal stromal cells drive significant biological changes in human breast cancer cells Školeková S., Kučerová L., Kozovská Z. Ústav experimentálnej onkológie SAV BACKGROUND: The discovery that mesenchymal stromal cells are recruited into tumor site where they can interact with tumor cells via direct contact or paracrine regulatory mechanism has led to increased interest in the function of MSCs in tumors. We have anayzed the influence of adipose tissue-derived mesenchymal stromal cells on SKBR3, MCF7 and MDA231 breast cancer cell line. METHODS: The tumor cells stably expressing green fluorescent protein were cultivated with adiposetissue mesenchymal stromal cells (AT-MSC) .We analysed chemosensitivity and proliferation by fluorimetric and mammosphere culture assay. The expression of specific genes was analysed by realtime PCR. RESULTS: We have observed AT-MSC promoted epithelial-to-mesenchymal transition in tumor cells and inhibition of the proliferation of EGFP-SKBR3 tumor cells. We have proposed that SDF-1α/CXCR4 signalling axis could be involved in AT-MSC mediated inhibition of tumor growth based on previous reports so we used AMD 3100, inhibitor of SDF-1α/CXCR4 signalling axis. Our data showed that blocking SDF-1α/CXCR4 signalling axis by AMD 3100 caused suppression of AT-MSC mediated inhibition effect on EGFPSKBR3 tumor growth. Morphological changes correlated also with molecular changes ? we have observed changed gene expression of EMT related genes in tumor cells cultivated with AT-MSC in comparison to unaffected parental cells. There was also changed expression of genes correlated with tumor growth, metastatic spread and angiogenesis. The EGFP-SKBR3 and EGFP-MCF7 cells cultivated with AT-MSCs showed also increased chemoresistance to chemotherapeutic drugs and increased sphere formation. CONCLUSION: In our work we report AT-MSC mediated changes in tumor cells proliferation, chemoresistance, gene expression and sphere formation. This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0230–11

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STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Intratékální aplikace lidských mesenchymálních stromálních buněk z kostní dřeně zlepšuje motoriku, zvyšuje přežívání a chrání perineuronální sítě potkanů s amyotrofickou laterální sklerózou Turnovcová K., Forostyak S., Homola A., Syková E., Jendelová P. Ústav experimentální medicíny AV ČR v. v. i. Amyotrofická laterální skleróza (ALS) je progresivní neurodegenerativní onemocnění postihující centrální a periferní motoneurony a postupně vedoucí k jejich zániku. Diagnóza je založena převážně na klinickém vyšetření, příčina je s výjimkou vzácných familiárních případů neznámá a neznámá je i prevence, spouštěcí faktory, terapie a mechanismy bránící dalšímu rozvoji. Buněčná terapie může být novou možností, jak ovlivnit rozvoj ALS neurotrofní podporou či přímo nahrazením dysfunkčních neuronů a gliových buněk. V této studii jsme se zabývali transplantací lidských mesenchymálních stromálních buněk (hMSC) do itratékálního prostoru u potkaního modelu amyotrofické laterální sklerózy. 500 000 hMSC izolovaných z kostní dřeně a resuspendovaných v 50 μl média bylo podáno cestou cisterna magna do intratékálního prostoru u symptomatických transgenních potkanů SOD1 G93A (n=11). SOD1 G93A kontrolám (n=9) bylo podáno pouze vehikulum. Zvířata byla testována behaviorálně (BBB test, síla úchopu, kontrola hmotnosti) a elektrofyziologicky (kondukční studie a počet motorických jednotek na m. gastrocnemius). U transplantovaných i kontrolních zvířat byly hodnoceny hladiny zánětlivých cytokinů v likvoru. Po ukončení pokusu bylo provedeno imunohistologické zhodnocení. Transgenní zvířata byla indikována k zákroku při objevení se prvních klinických symtomů. Přežívání transplantovaných zvířat bylo delší o 13,6 dne oproti kontrolám. Zvířata s buňkami vykazovala vyšší sílu úchopu a lepší hybnost v BBB testu než zvířata s podaným vehikulem. Rozdíly v motorické aktivitě se objevily bezprostředně po podání buněk. Elektrofyziologické studie prokázaly progresivní ztrátu počtu motorických jednotek na m. gastrocnemius, zatímco rychlost vedení nervem nebyla narušena. Kvantitativní analýza po barvení pomocí wisteria floribunda (WFA) prokázala signifikantně vyšší hustotu perineuronálních sítí (PNN) ve ventrálních rozích cervikální a lumbální míchy u zvířat léčených hMSC ve srovnání s SOD1 G93A kontrolami. Analýza cytokinů v likvoru prokázala zvýšení hladin MCP-1 a IL1α u SOD1 G93A zvířat léčených pomocí hMSC oproti ostatním sledovaným skupinám. Popsali jsme zde, že PNN jsou postiženy u SOD1 G93A potkanů a mohou sloužit jako diagnostický marker rozvoje choroby. Podání hMSC pomáhá ochránit PNN kolem míšních motoneuronů, zlepšuje motorickou aktivitu a prodlužuje délku přežití symptomatických zvířat, dále má přímý vliv na imunologické děje u postižených zvířat Lze předpokládat, že v budoucnu bude možné podání hMSC využít i v terapii ALS pacientů. Studie vznikla za podpory grantů GAČR P304/11/0189 a P304/12/G069.

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SBORNÍK ABSTRAKT Freezing of mesenchymal stem cells in different media Verdanova M.1,2,3, Broz A.1,2, Kalbacova M.1,4 Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, 128 08 Prague 2, Czech Republic1 Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague 2, Czech Republic2 J. Heyrovsky Institute of Physical Chemistry of the ASCR, v. v. i., Dolejškova 2155/3, 182 23 Prague 8, Czech Republic3 Biomedical Centre, Faculty of Medicine in Pilsen, Charles University, Czech Republic4 The aim of this study was to determine the influence of fetal bovine serum (FBS) on survival of the frozen human mesenchymal stem cells (hMSC). We also investigated usage of sericin (sticky protein created by silkworms) as the substitute for FBS in freezing medium. We determined cell viability by cell counting 24h after thawing and by measuring of cell ability to form colonies after 2 weeks. We observed that 1% sericin could serve as a replacement for FBS during freezing of cells. It is very useful to have suitable non-FBS freezing medium because of some difficulties connected with usage of FBS (non-human source, every batch is different). On the other hand, we also observed that FBS at all is not essential part of freezing medium in contrast to DMSO, which is essential for cell survival during freezing. Culture medium and 10% DMSO only is suitable freezing medium for hMSC.

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STEM CELLS IN 21 ST CENTURY: FROM RESEARCH TO MODERN CELL THERAPY Exprese MHC-I na buňkách vedlejší populace TRAMP-C2, myšího modelu karcinomu prostaty Žlabová A., Reiniš M. Ústav molekulární genetiky Úvod: Vedlejší populace (side population, SP) je subpopulace mnohých maligních nádorů či nádorových linií, zmiňovaná jako možný zdroj nádorových kmenových buněk (CSC), kterým bývají připisovány schopnosti založit nádor, metastázu, odolávat terapii či v neposlední řadě unikat imunitnímu dozoru. Jedním z obecně známých mechanismů úniku nádorových buněk imunitnímu dozoru je snížení MHC-I exprese. Buňky linie TRAMP-C2, myšího modelu karcinomu prostaty, mají částečně utlumenou povrchovou expresi MHC-I. Podle literatury 1–2% buněk linie TRAMP-C2 tvoří SP. Cílem naší práce bylo porovnat rozdíly v expresi MHC-I a parametry experimentálně indukované exprese MHC-I pomocí IFNγ v buňkách SP a v buňkách ostatních (nonSP) TRAMP-C2. Schéma práce: Buňky TRAMP-C2 byly před užitím k experimentům explantovány z nádoru myši C57BL/6 a několikrát in vitro pasážovány pro stabilizaci linie. Analýzou dat z průtokové cytometrie po inkubaci s Hoechst 33342 a fluorescenčně značenou protilátkou proti MHC-I byla znázorněna SP a povrchová exprese MHC-I. Poté byly vysortovány buňky SP a nonSP k analýze mRNA genů MHC-I-APM a genů interferonové signalizační dráhy metodou qPCR. V dalším experimentu byly buňky TRAMP-C2 vystaveny in vitro působení IFN-γ 100UI/ml po dobu 12, 24 a 48hod. Po inkubaci byla analyzována povrchová exprese MHC-I pomocí průtokové cytometrie. Výsledky: Ukázalo se, že buňky SP buněk TRAMP-C2 mají sníženou povrchovou expresi MHC-I oproti nonSP, pokles je patrný již na úrovni exprese mRNA genů mašinérie prezentující antigen v kontextu MHC-I (TAP1, TAP2, tapasinu, LMP1 a LMP7) a některých genů interferonové signalizační dráhy (STAT1, IRF1). Působením IFN-γ stoupá exprese MHC-I na povrchu SP i nonSP, avšak i po 48 hodinách je exprese MHC-I na povrchu buněk SP oproti nonSP výrazně nižší. Závěr: Tato práce ukazuje na modelu TRAMP-C2 signifikantně nižší expresi MHC-I na povrchu SP oproti nonSP. Výsledky experimentu s IFN-γ dokládají, že útlum exprese je na SP i nonSP reverzibilní, avšak i po 48 hodinách působení IFN-γ přetrvávají signifikantní rozdíly mezi SP a nonSP. Před případným zobecněním fenoménu nižší exprese MHC-I na buňkách SP oproti nonSP je však nutno jeho platnost ověřit na dalších buněčných liniích.

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